Treatment of sarcoma with interleukin 1α polypeptides

ABSTRACT

DNAs encoding polypeptides having interleukin 1, vectors inserted with said DNAs, hosts transformed with said recombinant vectors, polypeptides having interleukin 1 activity which are produced by cultivation of the transformed hosts, derivatives of the polypeptides, pharmaceutical composition containing said polypeptides or derivatives, their use as an antitumor or antiinfectious agent, and processes for the preparation thereof.

This is a continuation of copending application(s) Ser. No. 496,800filed on Mar. 21, 1990, now abandoned. Which is a divisional of Ser. No.812,796 filed Dec. 23, 1985 abandoned.

This invention relates to a DNA encoding a polypeptide havinginterleukin 1 activity, a vector having said DNA inserted thereinto, ahost transformed with said vector, a polypeptide having interleukin 1activity which is produced by cultivation of the transformed host,derivatives of the polypeptide, a pharmaceutical composition containingsaid polypeptide or derivative, their use as an antitumor orantiinfectious agent, and processes for the preparation thereof.

PRIOR ART

Gery et al., demonstrated the presence of a substance which augments themouse thymocyte proliferation by mitogen in the culture medium ofmacrophage, which has been called "lymphocytes activating factor" (LAF).Since 1979, it has been designated as "interleukin 1" (abbreviated as"IL-1"), and hence, in this specification, it is called as interleukin1.

It is known that IL-1 promotes proliferation of T and B lymphocytes andfurther acts on T lymphocyte and thereby promotes the production oflymphokines, particularly interleukin 2 (T lymphocyte proliferationfactor), and hence it is one of the important factors which take a roleon the production of antibody and the control of cellular immunity [cf.Staruch, M. J., et al., J. Immunol., 130, 2191 (1983)]. It is alsoreported that IL-1 is effective on the production of prostaglandin E orcollagenase, proliferation of fibroblast, and enhancement of naturalkiller (NK) cell activation by interleukin 2 or interferon [Simon, P.L., et al., "Lymphokines" Vol. 6, 47 (1982), Academic Press Inc.].

Thus, IL-1 participates not only immune response but also protective andrepairing mechanisms in vivo, and hence is expected to be used as amedicament.

IL-1 has been produced by culturing macrophage, peripheral mononuclearcells, macrophage-like cells (e.g. mouse P388D₁ cells), or monocytic ormyelocytic leukemia cells in the presence of an appropriate inducer andthen isolating from the culture medium, but the structure of the IL-1has not been clear.

It has been reported that human IL-1 isolated from the culture medium ofhuman monocytic leukemia cells (e.g. U937 cells) or human peripheralmononuclear cells has a molecular weight of 11,300 and 15,000 daltons[Mizel, S. B., et al., J. Immunol., 131, 1834 (1983); Schmidt, J. A., J.Exp. Med., 160, 772 (1984)].

It has recently been reported that cDNA encoding mouse IL-1 polypeptidehas been cloned from mouse P388D₁ cells and mouse IL-1 polypeptideconsisting of 156 amino acids has been produced in Escherichia coli[Lomedico, P. T., et al., Nature, 312, 458 (1984)]. It has also beenreported that cDNA encoding human IL-1 polypeptide has been cloned fromhuman mononuclear cells and the expression thereof has been succeeded[Auron, P. E., et al., Proc. Natl. Acad. Sci. USA, 81, 7907 (1984)].

However, the polypeptides and DNAs encoding them of this invention areclearly different from the above known products to distinguish IL-1 ofthe present invention from those known in the prior art, the instantIL-1 shall be designated IL-1a. Hereinafter, all occurrences of IL-1herein shall pertain to IL-1a.

There is no report as to rabbit IL-1 polypeptide and DNA encoding it.

BRIEF SUMMARY OF THE INVENTION

The present inventors have intensively studied on the production of IL-1by genetic engineering technique and have suceeded in cloning DNAsencoding human IL-1 and rabbit IL-1 and in the transformation of a hostwith an expression vector containing the cloned DNA and have confirmedthat the transformed host can produce IL-1 or a substance having IL-1activity.

That is, the present inventors have succeeded in the production of apolypeptide having human or rabbit IL-1 activity by cultivating humancells which have been differentiated into macrophage-like cells orrabbit macrophages in the presence of an inducer(s) suitable forproducing IL-1 and thereby accumulating IL-1 mRNA in the cells,preparing a cDNA library by using the mRNA as a template, cloning a DNAencoding human or rabbit IL-1 therefrom, determining whole nucleotidesequence of said DNA, and inserting the cloned DNA into an expressionvector, and then transforming a host with the vector to give atransformed host which can produce the desired polypeptide having IL-1activity. In the course of the study, the present inventors have foundthat the human or rabbit IL-1 is formed as a precursor and that thepolypeptide consisting of 159 amino acids at the C-terminus of saidhuman IL-1 precursor has potent IL-1 activity with molecular weight of18,000 and pI 5.3. Based on such findings, the present inventors havespecified that this polypeptide is mature human IL-1 and further thatmature rabbit IL-1 is a polypeptide consisting of 155 amino acids at theC-terminus of the rabbit IL-1 precursor by judging from the homology.

In this invention, the polypeptide consisting of 159 amino acids at theC-terminus of said human IL-1 precursor and the polypeptide consistingof 155 amino acids at the C-terminus of said rabbit IL-1 procursor arereferred to as a mature human IL-1 and a mature rabbit IL-1,respectively.

Moreover, it has been found that mature polypeptides having less aminoacid sequences than the sequences of the mature human IL-1 polypeptideconsisting of 159 amino acid residues show also IL-1 activity likemature human IL-1 polypeptide. Besides, high homologies have been foundbetween the nucleotide sequences coding for human and rabbit IL-1polypeptides, and between the deduced amino acid sequences of human andrabbit IL-1 polypeptides. It may be suggested therefrom that human andrabbit IL-1 polypeptides are phylogenetically derived from the samegene, and that a considerable portion(s) of the common regions are asequence(s) necessary for their biological activities.

An object of the invention is to provide a DNA encoding a polypeptidehaving IL-1 activity. Another object of the invention is to provide avector inserted with the DNA as mentioned above. Another object of theinvention is to provide a host transformed with said recombinant vector.A further object of the invention is to provide a polypeptide havingIL-1 activity which is produced by cultivation of the transformed hostas set forth above. A still further object of the invention is toprovide a derivative (substance) obtained from the polypeptide. A stillfurther object of the invention is to provide a pharmaceuticalcomposition containing said polypeptide or derivative as an activeingredient. A more further object of the invention is to provide aprocess for preparing the DNA, vector, transformed host and polypeptideor derivative. These and other objects and advantages of the inventionwill be apparent to person skilled in the art from the followingdescription.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and B show the test result of hybridization translation incloning of cDNA of rabbit IL-1, wherein the dotted line means ³H-thymidine incorporation in the control.

FIG. 2 shows the structure of human IL-1 chromosomal gene and itsrestriction endonuclease mapping (in Example 3).

FIGS. 3-5 show the steps of construction of expression vector pHLP101(in Example 4).

FIG. 6 shows SDS-polyacrylamide gel electrophoretic pattern of IL-1(209) obtained in Example 4.

FIG. 7 shows the steps of construction of the expression vector pHLP383(in Example 5).

FIG. 8 shows the steps of construction of the expression vector pHLP385(in Example 7).

FIG. 9 shows the steps of construction of the expression vector pHLP373(in Example 10.

FIG. 10 shows the steps of construction of the expression vector pRLP383(in Example 15).

FIG. 11 shows the nucleotide sequence and the corresponding amino acidsequence from the coding region of the human IL-1 gene and is the sameas Table 5.

FIG. 12 shows the nucleotide sequence and the corresponding amino acidsequence from the coding region of the rabbit IL-1 gene and is the sameas Table 7.

DETAILED DESCRIPTION OF THE INVENTION

For simplification of the description, the following abbreviations areused in the present specification and claims.

    ______________________________________                                        A:             adenine                                                        C:             cytosine                                                       G:             guanine                                                        T:             thymine                                                        Ala:           alanine                                                        Arg:           arginine                                                       Asn:           asparagine                                                     Asp:           aspartic acid                                                  Cys:           cysteine                                                       Gln:           glutamine                                                      Glu:           glutamic acid                                                  Gly:           glycine                                                        His:           histidine                                                      Ile:           isoleucine                                                     Leu:           leucine                                                        Lys:           lysine                                                         Met:           methionine                                                     Phe:           phenylalanine                                                  Pro:           proline                                                        Ser:           serine                                                         Thr:           threonine                                                      Trp:           tryptophan                                                     Tyr:           tyrosine                                                       Val:           valine                                                         DNA:           deoxyribonucleic acid                                          cDNA:          complementary DNA                                              sscDNA:        single-stranded cDNA                                           dscDNA:        double-stranded cDNA                                           RNA:           ribonucleic acid                                               mRNA:          messenger RNA                                                  poly(A)mRNA:   poly(A)-containing mRNA                                        dATP:          deoxyadenosine triphosphate                                    dCTP:          deoxycytidine triphosphate                                     dGTP:          deoxyguanosine triphosphate                                    dTTP:          deoxythymidine triphosphate                                    oligo(dC):     oligodeoxycytidylic acid                                       oligo(dG):     oligodeoxguanylic acid                                         oligo(dT):     oligodeoxythymidylic acid                                      poly(A):       polyadenylic acid                                              polv(U):       polyuridylic acid                                              poly(dC):      polydeoxycytidylic acid                                        poly(dG):      polydeoxyguanylic acid                                         ATP:           adenosine triphosphate                                         EDTA:          ethylenediaminetetraacetic acid                                kb:            kilobases                                                      kbp:           kilobase pairs                                                 bp:            base pairs                                                     ______________________________________                                    

In the present specification and claims, the nucleotide sequence shownby a single strand is the nucleotide sequence of a sense strand, and theleft end is a 5'-terminus and the right end is a 3'-terminus. In theamino acid sequence, the left end is an N-terminus, and the right end isa C-terminus.

[I] DNAs

(A) Designation of DNAs

The DNAs of this invention are DNAs having or containing a nucleotidesequence corresponding to an amino acid sequence represented by theformula [I-1] shown in the attached Table 1-1, wherein one to sixteen,preferably one to fifteen, amino acid residues at the N-terminus and/orone to seven, preferably one to five, amino acid residues at theC-terminus may be deleted, and DNAs having or containing a nucleotidesequence corresponding to an amino acid sequence represented by saidformula [I-1] which has, at the N-terminus thereof, further an aminoacid sequence represented by the formula [I-2] shown in the attachedTable 2-1, wherein an amino acid residue(s) at the N-terminus of theamino acid sequence of the formula [I-2] may be deleted.

These DNAs are ones derived from human gene or cDNA coding for humanIL-1.

A DNA consisting of a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] codes for mature human IL-1,and a DNA consisting of the above nucleotide sequence combined at the5'- terminus thereof with a nucleotide sequence corresponding to anamino acid sequence represented by the formula [I-2] codes for humanIL-1 precursor.

The DNAs of this invention includes also DNAs having or containing anucleotide sequence corresponding to an amino acid sequence representedby the formula [A-1] shown in the attached Table 3-1, which may furtherhave at the N-terminus thereof an amino acid sequence represented by theformula [A-2] shown in the attached Table 4-1, wherein an amino acidresidue(s) at the N-terminus of said amino acid sequence of the formula[A-2] may be deleted.

These DNAs are ones derived from rabbit gene or cDNA coding for rabbitIL-1. A DNA consisting of a nucleotide sequence corresponding to anamino acid sequence represented by the formula [A-1] codes for maturerabbit IL-1, and a DNA consisting of the above nucleotide sequencecombined at the 5'-terminus thereof with a nucleotide sequencecorresponding to an amino acid sequence represented by the formula [A-2]codes for rabbit IL-1 precursor.

Preferred DNAs of this invention are

(i) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1],

(ii) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which two amino acidresidues at the N-terminus are deleted,

(iii) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which ten amino acidresidues at the N-terminus are deleted,

(iv) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which fourteen or fifteenamino acid residues at the N-terminus are deleted,

(v) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which four amino acidresidues at the C-terminus are deleted,

(vi) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which five amino acidresidues at the C-terminus are deleted, and

(vii) a DNA having a nucleotide sequence corresponding to an amino acidsequence represented by the formula [I-1] in which fifteen amino acidresidues at the N-terminus and four amino acid residues at theC-terminus are deleted.

Further, a DNA having a nucleotide sequence corresponding to an aminoacid sequence represented by the formula [A-1] is also included as apreferred DNA of this invention.

Above DNAs may have an initiation codon at 5'-terminus and/or atermination codon(s) at the 3'-terminus.

The typical nucleotide sequences corresponding to amino acid sequencesrepresented by the formulae [I-1] and [A-1] are nucleotide sequencesrepresented by the formulae [II-1] and [B-1] shown in Table 1-2 and 3-2,respectively.

The typical nucleotide sequences corresponding to amino acid sequencesrepresented by the formulae [I-2] and [A-2] are nucleotide sequencesrepresented by the formulae [II-2] and [B-2] shown in Tables 2-2 and4-2, respectively.

It should be understood that the DNAs of this invention include thefollowing DNAs:

a DNA encoding a polypeptide possessing IL-1 activity,

a DNA encoding a polypeptide possessing IL-1 activity which is derivedfrom human or rabbit IL-1 gene or cDNA,

a cDNA encoding mature human or rabbit IL-1 polypeptide or itsprecursor,

a DNA encoding mature human or rabbit IL-1 polypeptide which is preparedfrom human or rabbit genomic library,

a DNA resulting from chemical or enzymatical modification of a DNAencoding mature human IL-1 polypeptide,

a partially or fully synthesized DNA which is substantially the same asthe DNA encoding mature human or rabbit IL-1 polypeptide,

a DNA with degenerative codons encoding mature human IL-1 polypeptide,its modified polypeptide or rabbit IL-1 polypeptide,

a DNA encoding mature human IL-1 polypeptide, its modified polypeptideor rabbit IL-1 polypeptide and having an initiation codon and/or atermination codon(s) and/or a promoter followed by Shine-Dalgarnosequence upstream of the initiation codon,

a DNA containing at least a nucleotide sequence(s) which has a highlyhomologous portion(s) between the nucleotide sequence encoding humanIL-1 polypeptide and the nucleotide sequence encoding rabbit IL-1polypeptide, and

a DNA resulting from deletion of a part of 5'-terminal region from anucleotide sequence encoding human or rabbit IL-1 precursor polypeptide.

(B) Production of DNAs

Processes for the production of the DNAs of the invention will bedescribed hereinbelow.

According to this invention, the DNAs encoding human IL-1 polypeptideand rabbit IL-1 polypeptide can be produced by cultivating human andrabbit macrophages (or macrophage-like cells) together with aninducer(s), separating a fraction containing IL-1 mRNA from the inducedcells, preparing a cDNA library from the fraction, and cloning the IL-1cDNA from the cDNA library.

That is, it can be produced through the following steps:

a) cultivating macrophage or macrophge-like cells together with aninducer(s),

b) separating a fraction containing IL-1 mRNA from the induced cells,

c) preparing a single-stranded cDNA (sscDNA) from the mRNA by using areverse transcriptase and then converting it to a double-stranded cDNA(dscDNA),

d) inserting the dscDNA into a vector,

e) introducing the recombinant vector into a host to transform it andconstruct a cDNA (colony) library,

f) cloning a cDNA encoding IL-1 polypeptide from the library which isconstructed by the steps a) to e).

If desired, modification (as step g) of the DNA produced as above cangive other DNAs of this invention.

On the other hand, a DNA encoding human or rabbit IL-1 polypeptide canalso be obtained by cloning a DNA coding for human or rabbit IL-1polypeptide from the genomic library (e.g. DNA fragments inserted intobacteriophage Charon 4A), followed by removing intron regions.

The processes for producing the cDNA of this invention will be describedin more detail as to the DNA encoding human IL-1 polypeptide or itsmodified polypeptide. The processes for production of the DNA encodingrabbit IL-1 polypeptide are almost the same as the case of human IL-1polypeptide.

The operations and conditions in the individual step of the processes tobe described hereinbelow are well known in the art, and the processes ofthis invention are never limited to these specific procedures.

(1) Preparation of human IL-1 mRNA

Step a: Human IL-1 mRNA can be obtained, for example from human leukemiacells by the following method.

Human leukemia cells are seeded at a cell density of 1×10⁵ to 5×10⁶cells/ml, and they are cultivated with an inducer(s) for differentiationinto macrophage-like cells. The amount of the inducer(s) variesdepending upon its type, kind of the leukemia cells, conditions ofcultivation and so on. In the case of using the inducer(s) as describedbelow, generally its final concentration is preferably about 100 to2,000 ng/ml. The cultivation is carried out at 35° to 38° C., preferablyabout 37° C. in a humidity of about 90 to 100% in air containing about 5to 10% of carbon dioxide for about 24 to 72 hours.

Human leukemia cells which can be used for the above step are all ofhuman leukemia cells which are differentiated into macrophage-like cellsby stimulating with the inducer. Examples include HL-60 cell (ATCC,CCL240), THP-1 cell, Mono-1-207 cell, and primary cells obtained frompatients with leukemia.

As an inducer for differentiation, diterpenes such as phorbol esters,mezerein and retinoic acid can be used.

Various culture media suitable for the cultivation of mammalian cellscan be used as the culture medium. Examples include RPMI-1640, Eagle'sMEM medium and Dulbecco's modified MEM medium [for the compositions ofthe above media, see for example, "Cell Cultivation Manual" edited by Y.Sohmura, Kodansha (1982); and J. Paul "Cell and Tissue Culture", E. & S.Livingstone Ltd. (1970)]. Preferably, animal serum, such as fetal bovineserum or calf serum, is added to the culture medium in an amount ofabout 1 to 20%.

The following experiments will be made after confirming that leukemiacells are differentiated into macrophage-like cells, and get adherent toa dish.

However, in the case of using human macrophages collected from lung,blood, abdomen, placenta, spleen and other tissue, the differentiationstep as mentioned above can be omitted.

Step b: After the cultivation as above, the culture medium andnon-adherent cells are removed by suction. Then, the medium containingan inducer(s) for synthesis of IL-1 (for example, endotoxin derived froma gram-negative bacterium, a diterpene such as phorbol esters andmezerein) and a protein synthesis inhibitor (for example, cycloheximide)is added into the dish, and the cultivation is further continued for 3to 8 hours to accumulate IL-1 mRNA in the macrophages. The amount of theendotoxin is generally about 0.1 to 1000 micrograms/ml (finalconcentration; the same as hereinbelow), preferably about 1 to 100micrograms/ml. The amount of a phorbol ester is about 1 to 2000 ng/ml.The amount of cycloheximide is preferably 0.1 to 50 micrograms/ml.

After the cultivation, total RNA is extracted from the cells by acustomary method, for example the method of Chirgwin et al.[Biochemistry, 18, 5294 (1979)], and then by affinity columnchromatography on oligo(dT)-cellulose or poly(U)-Sepharose, or by abatch method, a fraction containing poly(A)mRNA is separated. Anenriched mRNA fraction with human IL-1 mRNA can be obtained by acid-ureaagarose gel electrophoresis or sucrose density gradient centrifugationof the poly(A)mRNA fraction.

To confirm that the resulting mPNA fraction is the desired onecontaining mRNA encoding human IL-1 polypeptide, the mRNA is made totranslate into a protein and its biological activity is examined. Thiscan be carried out, for example, by injection it into the oocytes ofXenopus laevis or by applying it to a suitable protein synthesizingsystem, such as reticulocyte lysate or wheat germ cell-free proteinsynthesizing system and by confirming that the translated protein hasIL-1 activity.

(2) Cloning of Human IL-1 cDNA

Step c The mRNA fraction obtained in the above (1) is used as a templateand an oligo(dT) is used as a primer in order to synthesize a sscDNA byusing reverse transcriptase (for example, that derived from avianmyeloblastosis virus (AMV)) in the presence of dATP, dGTP, dCTP anddTTP. Then, the sscDNA is used as a template, and a dscDNA issynthesized by using reverse transcriptase on E. coli DNA polymerase I(large fragment).

Steps d and e: The resulting dscDNA is inserted, for example, into therestrictor endonuclease PstI cleavage site of plasmid pBR322 by aconventional method, for example the poly(dG)-poly(dC) homopolymerextension method (Nelson, T. S. "Methods in Enzymology", 68, 41 (1979),Academic Press Inc., New York). The resulting recombinant plasmids areintroduced into a host such as E. coli X1776 in accordance with themethod of Cohen et al. [Proc. Natl. Acad. Sci., USA, 69, 2110 (1972)] totransform it, and by selecting tetracycline-resistant colonies, a cDNA(colony) library is prepared.

Step f: To select the desired clones harboring recombinant plasmidscontaining a cDNA insert encoding human IL-1 polypeptide, the followingmethods will be used.

If a suitable DNA fragment, for example a cDNA fragment or a chemicallysynthesized DNA fragment corresponding to an animal (but not human) IL-1polypeptide, can be previously obtained, the above cDNA library issubjected to colony hybridization assay [Hanahan, D., et al., Gene, 10,63 (1980)] by using the above DNA fragment labelled with ³² P as aprobe, and the desired clones harboring recombinant plasmids containinga cDNA insert having a nucleotide sequence(s) complementary to the DNAprobe used are selected.

If such a suitable IL-1 DNA probe as above cannot be obtained, thedesired clones are screened by colony hybridization using induction-plusand induction-minus probes and by mRNA hybridization-translation assay,for example according to the procedures as described below.

A ³² P-labelled cDNA is synthesized using the mRNA fraction containinghuman IL-1 mRNA obtained in the above (1) as a template and used as aninduction-plus probe. Separately, by using as a template a mRNA fractionobtained by the same procedure as described in the above (1) except thatthe non-induced macrophages are used as a starting material, a ³²P-labelled cDNA is synthesized. This ³² P-labelled cDNA is used as aninduction-minus probe. From the above cDNA library, there are selectedplasmid clones which are strongly hybridized with the induction-plusprobe, but not hybridized with the induction-minus probe.

The A mRNA hybridization-translation assay is carried out in order toconfirm that the selected clones harbor a cDNA insert encoding humanIL-1 polypeptide. The plasmid DNAs are isolated from the above selectedclones, converted into a single-stranded DNA by heating or alkalitreatment, and fixed onto nitrocellulose filters. The mRNA fractioncontaining human IL-1 mRNA obtained according to the method as mentionedin the above (1), is added to the filters to hybridize with the fixedDNA. Then, the hybridized mRNA is eluted and recovered. The recoveredmRNA is injected into oocytes of Xenopus laevis to determine whether therecovered mRNA encodes human IL-1 polypeptide.

The above methods give transformants harboring a recombinant plasmidhaving a cDNA fragment containing a nucleotide sequence complementary tothe human IL-1 mRNA.

When the obtained cloned cDNAs do not contain whole coding region ofhuman IL-1 polypeptide, cDNAs of a larger size are selected by screeningthe cDNA library using as a probe the cloned cDNA fragment from thetransformant selected as above.

The cloned cDNA encoding a polypeptide containing the amino acidsequence of human IL-1 polypeptide can be proved finally by analyzingthe nucleotide sequence of some of the resulting cloned cDNA fragmentsin accordance with, for example the Maxam-Gilbert method [Proc. Natl.Acad. Sci., USA, 74, 560 (1977)] or the dideoxy method using a M13 phage[Sanger, F., et al., Proc. Natl. Acad. Sci., USA, 74, 5463 (1977), andMessing, J., Methods in Enzymology, 101, 20 (1983)].

Step g (Modification): If desired, the cloned cDNA obtained as above canbe modified by techniques known per se to form the DNA having and/orcontaining a nuclectide sequence deleted one or more codons and/orreplaced with other codon(s) or degenerative codon(s).

Modification is carried out, for example, by cleaving the DNA withsuitable restriction endonuclease(s) and splitting off one or morecodons with suitable exoncleases and/or endonucleases singly or incombination, followed by replacing with degenerative condons, forexample, those synthesized chemically by the phosphotriester method[Ohtsuka, E., et al., Heterocycles, 15, 395 (1981)], or by ligationwithout any supplement of codons to prepare DNA having one or morecodons deleted.

[II] Polypeptides

The DNAs prepared in the above [I] are adequately inserted into anexpression vector, and a host is transformed with said expressionvector. Thereafter, the transformed host is cultivated to produce thedesired polypeptide.

(A) Designation of polypeptides

The polypeptide produced by using a DNA derived from human IL-1 gene orcDNA is a polypeptide having an amino acid sequence represented by theformula [I-1] shown in the attached Table 1-1, wherein one to sixteen,preferably one to fifteen, amino acid residues at the N-terminus and/orone to seven, preferably one to five, amino acid residues at theC-terminus may be deleted.

Besides, the polypeptide produced by using a DNA derived from rabbitIL-1 gene or cDNA is a polypeptide having an amino acid sequencerepresented by the formula [A-1] show in the attached Table 3-1.

Preferred polypeptides of this invention are as follows:

(1) a polypeptide having an amino acid sequence represented by theformula [I-1],

(2) a polypeptide having an amino acid sequence represented by theformula [I-1] in which two amino acid residues at the N-terminus aredeleted,

(3) a polypeptide having an amino acid sequence represented by theformula [I-1] in which ten amino acid residues at the N-terminus aredeleted.

(4) a polypeptide having an amino acid sequence represented by theformula [I-1] in which fourteen or fifteen amino acid residues at theN-terminus are deleted,

(5) a polypeptide having an amino acid sequence represented by theformula [I-1] in which four amino acid residues at the C-terminus aredeleted,

(6) a polypeptide having an amino acid sequence represented by theformula [I-1] in which five amino acid residues at the C-terminus aredeleted,

(7) a polypeptide having an amino acid sequence represented by theformula [I-1] in which fifteen amino acid residues at the N-terminus andfour amino acid residues at the C-terminus are deleted, and

(8) a polypeptide having an amino acid sequence represented by theformula [A-1].

It should be understood that the polypeptides of this invention includethe following polypeptides:

a polypeptide possessing IL-1 activity,

a polypeptide possessing IL-1 activity which is derived from human orrabbit IL-1 gene or cDNA,

a polypeptide produced by cultivating a host transformed with anexpression vector having the DNA of this invention inserted hereinto,

a human or rabbit IL-1 polypeptide,

a modified polypeptide of human IL-1 polypeptide,

a degrated substance of human or rabbit IL-1 polypeptide in the host,

a polypeptide which has at least a highly homologous portion(s) betweenhuman IL-1 polypeptide and rabbit IL-1 polypeptide.

(B) Production of Polypeptides

Processes for the production of the polypeptides of this invention willbe described hereinbelow.

According to this invention, a human IL-1 polypeptide or its modifiedpolypeptide and a rabbit IL-1 polypeptide (hereinafter, whole of thesepolypeptides are occasionally referred to merely as "the polypeptide")can be produced by the following steps:

i) inserting a DNA having or containing a nucleotide sequence encodingthe polypeptide into an expression vector,

ii) introducing the recombinant vector into a host,

iii) cultivating the host transformed with the recombinant vector toproduce the polypeptide,

iv) collecting the cultured cells and extracting the polypeptideproduced from them, and

v) purifying the polypeptide by conventional purifying methods forproteins.

If desired, the polypeptide produced through the above steps may bemodifed (as step vi) to produce other polypeptides of this invention, ortheir derivatives or salts.

A detailed description will follow of the processes for producing thepolypeptide by using a DNA encoding the polypeptide.

Step i An expression vector for production of the polypeptide can beobtained by inserting a cloned DNA encoding the polypeptide. All vectorswhich proliferate in microorganisms to be transformed can be used.Examples include plasmids (such as E. coli plasmid pBR322), phages (suchas lambda phage derivatives), and viruses (such as SV40). They may beused singly or in combination, for example as a pBR322-SV40 hybridplasmid. The site of insertion of the DNA can be properly selected. Inother words, a suitable site of a suitable vector may be cleaved with asuitable restriction endonuclease(s) in a customary manner, and thecloned DNA of suitable length may be inserted into the cleavage site.

More specifically, an expression vector for production of thepolypeptide (non-fused) is constructed by joining a DNA fragmentcontaining the nucleotide sequence encoding the polypeptide in which theinitiation codon ATG is added to the 5'-terminus and the terminationcodon(s) (TAA, TAG or TGA) is added to the 3'-terminus, down stream aDNA fragment with a suitable promoter and the Shine-Dalgarno sequenceand inserting it into a vector. An expression vector for the productionof the fused polypeptide may be constructed by inserting the DNAfragment having the nucleotide sequence encoding the polypeptide inwhich the termination codon is added to its 3'-terminus, into the vectorso that the translational reading frame coincides with that of thestructure gene to be fused.

Examples of the promoters are lac, trp, tac, phoS, phoA, P_(L) and SV40early promoters.

Step ii Transformants are obtained by introducing the expression vectorinto a host such as microorganism, animal or plant cell. For example, E.coli is transformed by the method of Cohen et al. [Proc. Natl. Acad.Sci., USA, 69, 2110 (1972)]. Then, by cultivating the transformant, thepolypeptide or the polypeptide with a methionine at its N-terminus isproduced. The product can be accumulated either in the cytoplasm or inthe periplasm of the host cell depending upon the method of constructingthe expression vector. To cause the polypeptide to be secreted in theperiplasm, one can construct an expression vector by using a gene codingfor a secretory protein, such as an alkaline phosphatase gene (phoA) ora phosphate binding protein gene (phoS), and joining the DNA encodingthe polypeptide in the correct translational reading frame into theabove gene at a suitable site following a DNA region encoding the signalpeptide.

Steps iii and iv The resulting transformant is cultivated under suitableconditions for the transformant until the polypeptide desired is fullyproduced. Then, the polypeptide is extracted from the culture. When theproduced polypeptide is accumulated in the cytoplasm, the host cells aredestroyed by lysozyme digestion and freezing and thawing or sonicationor by using a French press, and then centrifuged or filtered to collectthe extract. When it is accumulated in the periplasm, it can beextracted, for example, by the method of Willsky et al. [J. Bacteriol.,127, 595 (1976)].

Step v The polypeptide so obtained can be purified by conventionalpurifying methods for proteins, for example by combinations of saltingout, ultrafiltration, dialysis, ion exchange chromatography, gelfiltration, electrophoresis, affinity chromatography, etc.

By the foregoing processes, the polypeptide and/or the polypeptide witha methionine at the N-terminus of the polypeptide can be produced.

[III] Modification of polypeptides

Substances resulting from modification of the polypeptide includepolypeptides resulting from addition of an amino acid or a peptide(consisting of two or more amino acids) to the N-terminus and/orC-terminus of the polypeptide; polypeptides resulting from deletion ofone or more amino acids from the polypeptide; polypeptides resultingfrom change of asparagine or glutamine residue into aspartic acid orglutamic acid residue, respectively, or from change of aspartic acid orglutamic acid residue into asparagine or glutamine residue,respectively, and further include derivatives of the polypeptides, suchas esters, acyl derivatives or acid amides, which are formed by using afunctional group in the molecule, an amino residue of the N-terminus ora carboxy residue of the C-terminus, and their salts formed by usingamino residues or carboxy residues with, for example, sodium hydroxide,potassium hydroxide, arginine, caffein, procaine, hydrochloric acid,gluconic acid and so on.

Such a modification or preparation of derivatives of the polypeptide arecarried out by the techniques known per se, for example, according tothe method as described in "Chemical Modification of Proteins" by Means,G. E. and Feeney, R. E., Holden-Day, Inc., California (1971).

The polypeptide of this invention may exist as its aggregate, or mayalso be changed in the host or in the course of separation. In case thatcysteine residue is a component of a polypeptide of this invention, thisresidue may constitute S--S bond between the molecules. These changedpolypeptides are also included in the present invention.

In case of transformation of a host with a vector inserted with a DNAencoding human or rabbit IL-1 precursor or a DNA containing a regionupstream a nucleotide sequence encoding mature human or rabbit IL-1, thetransformed host may occasionally produce a precursor polypeptide, apolypeptide combined with a polypeptide corresponding to the upstreamDNA at the N-terminus of the mature polypeptide with or without themature polypeptide. These polypeptides are also included in thisinvention.

[IV] Chemical and Physical Properties of Polypeptides:

Chemical and physicochemical properties of mature human IL-1 polypeptideand its modified polypeptides will be described below.

(A) Procedures for analyses

Molecular weight

The molecular weight was measured by sodium dodecyl sulfate(SDS)-polyacrylamide gel (gel concentration; 12.5%) electrophoresis.

A sample solution was mixed with an equal volume of 0.125M Tris-HCl (pH6.8) buffer containing 4% SDS, 10% 2-mercaptoethanol, 20% glycerol and0.02% bromophenol blue, followed by standing for 30 minutes at roomtemperature. The mixture was subjected to SDS-polyacrylamide gelelectrophoresis under the conditions of 200 volts for 3 hours using 0.2Mglycine solution containing 25 mM Tris and 0.1% SDS as anelectrophoresis electrolyte. After electrophoresis, each migration lanewas separated by cutting, and a lane was stained with CoomassieBrilliant Blue C-250 to detect proteins. Then, another lane was cut intostrips of 2 mm-width, and each strip was immersed into RPMI-1640 mediumcontaining 5% fetal bovine serum or a 1% ammonium bicarbonate solutionto elute the polypeptide in the gel strip. The eluate from each gelstrip was subjected to IL-1 activity assay according to the method asmentioned in Example 1.

As molecular weight marker proteins, the following proteins were used;phosphorylase-b (MW: 94,000), bovine serum albumin (MW: 67,000),ovalbumin (MW: 43,000), carbonic anhydrase (MW: 30,000), soybean trypsininhibitor (MW: 20,100) and alpha-lactalbumin (MW: 14,400).

Determination of N-terminal amino acid and amino acid sequence

N-terminal amino acid was determined by dansylation method [Gray, W. R.,Methods in Enzymol., Vol XI, p139 (1967)].

The sample solution (100 microliters) was mixed with 10 microliters of10% SDS, 100 microliters of N-ethylmorpholine and 50 microliters ofdansyl chloride solution (5 mg/ml in acetone), and incubated at 37° C.for 1 hour. Then, 2 ml of acetone was added to the reaction mixture, andthe resulting precipitate was collected by centrifugation. After washingwith 80% acetone, the precipitate was dried in vacuo. And then it wasdissolved in 100 microliters of 6N HCl, and it was heated at 105° C. for5 to 18 hours in a glass tube sealed under the condition of vacuo. Theresulting dansylated amino acid was extracted by water-saturated ethylacetate from the sample dried in vacuo. The dansylated amino acid wasidentified by two dimentional thin layer chromatography using apolyamine sheet (Cheng-Chin Trading Co., Taiwan) according to the methodof Wood and Wang [Biochem. Biophys. Acta, 133, 369 (1967)].

N-terminal amino acid sequence was determined by the Edman degradationmethod [Arch. Biochem. Biophys., 22, 475 (1949)].

A phenylthiohydantoin-amino acid derived from an N-terminal amino acidby the Edman degradation method was identified by high-performanceliquid chromatography using a column of TSK-gel ODS-120A (Toyo SodaKogyo, Japan). These procedures were serially repeated to determine anewly formed N-terminal amino acid derivative sequentially.

Determination of C-terminal amino acid sequence

C-terminal amino acid sequence was determined by the enzymatic methodusing carboxypeptidases.

The sample was digested with carboxypeptidase-A and carboxypeptidase-Y.The free amino acids released were identified and determinedquantitatively by a micro-amino acid analyzer (Shimadzu Seisakusho,Japan) at appropriate intervals of the enzymatic digestion.

Isoelectric point

An isoelectric point was determined by isoelectro-focusing gelelectrophoresis at 5 watt for 3 hours using a 5% polyacrylamide flat gelwith a pH gradient created with Pharmalyte (Pharmacia, Sweden).

(B) Chemical and physiocochemical properties:

Chemical and physicochemical properties of mature human IL-1 polypeptideand its modified polypeptides were summarized in the following Tables(a), (b) and (c).

In Tables (a), (b) and (c), mature human IL-1 polypeptide and itsmodified polypeptides are as follows;

Mature human IL-1 polypeptide is a polypeptide obtained in Example 5-(2)which corresponds to the amino acid sequence from the amino acid No. 113to the amino acid No. 271 in Table 5.

IL-1(157) is a polypeptide obtained in Example 6-(2) which correspondsto the amino acid sequence from the amino acid No. 115 to the amino acidNo. 271 in Table 5.

IL-1(149) is a polypeptide obtained in Example 7-(2) which correspondsto the amino acid sequence from the amino acid No. 123 to the amino acidNo. 271 in Table 5.

IL-1(144) is a polypeptide obtained Example 8-(2) which corresponds tothe amino acid sequence from the amino acid No. 127 or 128 to the aminoacid No. 271 in Table 5.

IL-1(155-C) is a polypeptide obtained in Example 10-(2) whichcorresponds to the amino acid sequence from the amino acid No. 113 tothe amino acid No. 267 in Table 5.

                                      TABLE (a)                                   __________________________________________________________________________    Properties of mature human IL-1 polypeptide                                   item     mature human IL-1 polypeptide                                        __________________________________________________________________________    molecular weight                                                                       18,000 ± 500                                                      isoelectric point                                                                      pI 5.3 ± 0.3                                                      N-terminal amino                                                                       Ser--Ser--Pro--Phe--Ser--Phe--Leu--Ser--Asn--Val--                   acid sequence                                                                          Lys--Tyr--Asn--Phe--Met--Arg--Ile--Ile--Lys--Tyr--                            Glu--Phe--Ile--Leu--Asn--Asp--Ala--Leu--Asn--Gln--                   C-terminal amino                                                                       --Asx--Glx--Ala                                                      acid sequence                                                                 __________________________________________________________________________

                  TABLE (b)                                                       ______________________________________                                        Properties of IL-1(157), IL-1(149) and IL-1(144)                              item    IL-1(157)    IL-1(149)    IL-1(144)                                   ______________________________________                                        molecular                                                                             17,700 ± 300                                                                            16,100 ± 300                                                                            16500 ± 300                              weight                                                                        N-terminal                                                                            Pro--Phe--Ser--                                                                            Lys--Tyr--Asn--                                                                            Y--Ile--Ile--                               amino acid                                                                            Phe--,       Phe--Met--,  Lys--Tyr--,                                 C-terminal                                                                            From each of the above three polypeptide, the                         amino acid                                                                            following amino acids were released and                                       identified; Ala, Glx, Asx, Leu, Ile, Phe,                             ______________________________________                                    

                  TABLE (c)                                                       ______________________________________                                        Properties of IL-1(155-C)                                                     item           IL-1(155-C)                                                    ______________________________________                                        molecular      about 17,500                                                   weight                                                                        N-terminal     Ser--Ser--Pro--Phe--Ser--                                      amino acid                                                                    C-terminal     --Glx--Ile--Leu                                                amino acid                                                                    ______________________________________                                    

In the above tables, Glx represents Glu or Gln, Asx represents Asp orAsn, and Y represents Arg or Met-Arg.

[V] Formulation and use

For formulating the polypeptides or substances of this invention, theymay be in the form of a solution or a lyophilized product. From thestandpoint of long-term stability, they are desirably in the form oflyophylized products. It is preferred to add vehicles or stabilizers tothe preparations. Examples of the stabilizers include albumin, globulin,gelatin, protamine, protamine salts, glucose, galactose, xylose,mannitol, glucuronic acid, trehalose, dextran, hydroxyethyl starch, andnonionic surface-active agents (such as polyoxyethylene fatty acidesters, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenylethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethyleneglycerin fatty acid esters, polyoxyethylene hardened castor oil,polyoxyethylene castor oil, polyoxyethylene polyoxypropylene alkylethers, polyoxyethylene polyoxypropylene block copolymer, sorbitan fattyacid esters, sucrose fatty acid esters and glycerin fatty acid esters).

The polypeptides or substances of this invention are useful as anantitumor or antiinfectious agent because they regress tumorstransplanted in animals, and relieve animals from fatal microbialinfections.

Such preparations are preferably administered parenterally or topically.Parenteral routes such as intravenous, subcutaneous and intramuscularroutes are used when tumor cells or pathogens extend over a wide rangeof body, or when prevention of metastasis of tumors or microbialinfections is intended. Against local tumor tissues or local infections,local administration is preferred. The dosage varies depending upon thetype and size of tumors or infections, the condition of the patient andthe route of administration. An antiinflammatory agent such asindomethacin may be co-administered.

[VI] Examples

The following examples illustrate this invention more specifically. Itshould be understood however that the invention is in no way limited tothese examples.

For better understanding of the following examples, FIGS. 1 to 10 andTables 5-8 are attached to the present specification.

Example 1 Cloning and sequencing of cDNA encoding human IL-1

(1) Preparation of human IL-1 mRNA from human promyelocytic leukemiacells (HL-60)

HL-60 cells were seeded in Petri dishes (8 cm in diameter) at a celldensity of 1×10⁷ cells/dish in 10 ml of RPMI-1640 medium containing 10%fetal bovine serum with 500 ng/ml phorbol-12-myristate-13-acetate and500 ng/ml retinoic acid as a differentiation inducer. After cultivationat 37° C. in a fully humidified atmosphere containing 5% carbon dioxidefor 2 days, the culture medium and non-adherent cells were removed bysuction. Into the dishes to which the differentiated cells adhered, 10ml of RPMI-1640 medium containing 10% fetal bovine serum supplementedwith 10 micrograms/ml endotoxin (lipopolysaccharide derived from E.coli, which is hereinafter referred to as "LPS") as an inducer and 1microgram/ml cycloheximide (protein synthesis inhibitor) was added andfurther cultivated for 5 hours. The culture medium was removed bysuction, and the induced cells adhered to the dishes were lysed andhomogenized in a 6M guanidinium thiocyanate solution containing 0.5%sodium N-laurcyl sarcosinate, 5 mM sodium citrate and 0.1M2-mercaptoethanol. The homogenate was loaded on a 5.7M cesium chloridesolution containing 0.1M EDTA, and centrifuged for 20 hours at 26,500rpm using an ultracentrifuge (RPS27-2 rotor, Hitachi Koki, Japan) toobtain a total RNA fraction as pellets. The pellets were dissolved in asmall amount of a 7M urea solution containing 0.35M NaCl, 30 mM Tris-HCl(pH 7.4) and 20 mM EDTA, and recovered by precipitation from ethanol.From 1.5×10⁸ cells of HL-60, 1.7 mg of the total RNA was obtained. Thetotal RNA fraction was dissolved in 2 ml of 10 mM Tris-HCl (pH 7.4)buffer containing 1 mM EDTA (hereinafter, referred to as "TE solution"),and the solution was heated at 65° C. for 5 minutes. A NaCl solution wasadded to a final concentration of 0.5M, and the solution was appliedonto a column of oligo(dT)-cellulose previously equilibrated with the TEsolution containing 0.5M NaCl. Poly(A)mRNA was eluted from the columnwith the TE solution in an yield of about 75 micrograms.

The poly(A)mRNA was injected into the occytes of Xenopus laevis at adose of about 150 ng/oocyte, and the 10 oocytes were incubated in 100microliters of Barth's medium at 22° C. for 24 hours. The oocytes werehomogenized and centrifuged. The supernatant was subjected to the assayof IL-1 activity according to the method as mentioned below.

The RPMI-1640 medium containing 5% fetal bovine serum is used as amedium. A sample is diluted with the medium into an appropriateconcentration. Fifty microliters of the dilution is added into each wellof a 96 well multi-well plate (Flow Labs., USA). Furthermore, 50microliters of 50 micrograms/ml phytohemagglutinin-P (Difco Labs., USA)and 100 microliters of thymocyte suspension (1×10⁷ cell/ml) collectedfrom C3H/He (6-10 week old) mice are added into the well. The plate isincubated at 37° C. in a fully humidified atmosphere containing 5%carbon dioxide. After incubation for 2 days, 1 microcurie of ³H-thymidine is added into each well and the plate is further incubatedfor 18 hours. The thymocyte cells are collected onto a Harvester Filter(Flow Labs.) using a Titertek Cell Harvester (Flow Labs.). Theradioactivity of ³ H-thymidine incorporated into the cells is counted.The LAF (lymphocyte activating factor) activity is determined with theaugmentation of ³ H-thymidine incorporation by comparison with the ³H-thymidine incorporation of a control using the medium instead of atest sample.

The above method was used as an assay for the IL-1 activity in all ofthe following examples.

A 320-fold diluted solution of the supernatant prepared as above showedan incorporation of about 15,000-18,000 cpm of ³ H-thymidine. Itindicates that the poly(A)mRNA preparation contains IL-1 mRNA.

(2) Synthesis of cDNA

Complementary DNA was synthesized according to the method of Gubler andHoffman [Gene, 25, 263 (1983)] using the poly(A)mRNA obtained in theabove (1) as a template.

Six micrograms of the poly(A)mRNA were dissolved in 6 microliters ofdistilled water, and then thereto was added 0.6 microliter of 100 mMmethylmercury hydroxide solution. After standing at room temperature for10 minutes, 1.7 microliters of 500 mM 2-mercaptoethanol solutioncontaining 20 units of ribonuclease inhibitor (RNasin®, Product ofPromega Biotech, USA) was added to the mixture. After standing at roomtemperature for 5 minutes, 32 microliters of 50 mM Tris-HCl (pH 8.3)buffer containing 10 mM MgCl₂, 1.25 mM dGTP, 1.25 mM dATP, 1.25 mM dTTP,0.5 mM dCTP, 170 nM alpha-³² P-dCTP (specific radioactivity, 750Ci/mmole), 4 micrograms of oligo(dT)₁₂₋₁₈ and 120 units of AMV reversetranscriptase was added to the mixture and it was incubated at 42° C.for 60 minutes. The reaction was stopped by adding EDTA. The reactionmixture was extracted with phenol/chloroform (1:1), and ammonium acetatewas added to the aqueous phase to a final concentration of 2.5M. Theresulting sscDNA-mRNA hybrid was recovered from the aqueous phase byprecipitation from ethanol. The sscDNA-mRNA hybrid precipitate wasdissolved in 100 microliters of 20 mM Tris-HCl (pH 7.5) buffercontaining 5 mM MgCl₂, 10 mM (NH₄)₂ SO₄, 100 mM KCl, 0.15 mMbeta-nicotinamide-adenine dinucleotide, 5 micrograms of bovine serumalbumin, 0.04 mM of each of four deoxyribonucleotide triphosphates,dGTP, dATP, dTTP and dCTP, 1.25 units of E. coli ribonuclease H and 24units of E. coli DNA polymerase I. The reaction mixture was incubated at12° C. for 60 minutes, and then 2.5 units of E. coli DNA ligase wasadded, and it was further incubated at 22° C. for 60 minutes tosynthesize a dscDNA. The reaction was stopped by adding EDTA. The dscDNAwas extracted with phenol/chloroform and recovered by precipitation fromethanol as mentioned above.

(3) Preparation of oligo(dC)-tailed cDNA

The dscDNA obtained as above was dissolved in 100 microliters of 100 mMsodium cacodylate (pH 7.2) buffer containing 2 mM CoCl₂, 0.2 mMdithiothreitol, 0.1 mM alpha-³² P-dCTP (specific radioactivity, 1Ci/mmole) and 10 units of terminal deoxynucleotidyl transferase, andincubated at 37° C. for 30 minutes to permit the addition of oligo(dC)tails to the 3'-termini of dscDNA.

The reaction was stopped by adding EDTA. The oligo(dC)-tailed dscDNA wasextracted with phenol/chloroform, and recovered by precipitation fromethanol. The oligo(dC)-tailed dscDNA was dissolved in 10 mM Tris-HCl (pH7.4) buffer containing 1 mM EDTA and 100 mM NaCl so that it contained 2micrograms of the oligo(dC)-tailed dscDNA per ml.

(4) Construction of recombinant plasmids

Oligo(dC)-tailed pBR322 DNA (product of Bethesda Res. Labs. Inc., USA)and the oligo(dC)-tailed dscDNA obtained in the above (3) were dissolvedin 10 mM Tris-HCl (pH 7.4) buffer containing 1 mM EDTA and 100 mM NaClso that it contained 1.5 micrograms and 0.09 microgram, respectively, ina total volume of 1.5 ml.

The mixture was incubated sequentially at 65° C. for 10 minutes, at 57°C. for 2 hours and at 45° C. for 2 hours to perform annealing and toconstruct recombinant plasmids.

(5) Selection of transformants

E. coli X1776 strain was transformed with the recombinant plamidsobtained as above.

Specifically, E. coli X1776 was cultivated at 37° C. in 20 ml of L broth(composition: 10 g tryptone, 5 g yeast extract, 5 g NaCl and 1 g glucoseper liter, pH 7.2) supplemented with 100 micrograms/ml of diaminopimelicacid and 40 micrograms/ml of thymidine until the turbidity at 600 nmreached 0.5. The cells were collected by centrifugation at 4° C., andwashed with 10 ml of 10 mM Tris-HCl (pH 7.3) buffer containing 50 mMCaCl₂. The cells were resuspended in 2 ml of the same buffer as usedabove, and allowed to stand at 0° C. for 5 minutes. To 0.2 ml of thecell suspension was added 0.1 ml of the recombinant plasmid solutionobtained as above. The mixture was allowed to stand at 0° C. for 15minutes and then maintained at 42° C. for 2 minutes. Then, 0.5 ml of thesupplemented L broth as used above was added, and cultivation wascarried out with shaking for 1 hour. An aliquot of the culture wastaken, spread on the supplemented L broth agar plate containing 15micrograms/ml of tetracycline, and cultivated at 37° C. for about 12hours. A cDNA library was prepared by selecting transformants resistantto tetracycline.

(6) Cloning

Transformants harboring the recombinant plasmids containing cDNAsencoding human IL-1 were selected from the cDNA library obtained in theabove (5), by the colony hybridization assay using the cloned cDNAencoding rabbit IL-1 obtained in Example 2, as a probe.

Specifically, the cloned cDNA (about 1.1 kbp) encoding rabbit IL-1 wasisolated from the recombinant plasmid pRL15 as shown in Example 2-(6) bydigestion with a restriction endonuclease PstI, and labelled with ³² P.This labelled cDNA was used as a probe for screening the cDNA library toselect transformants having a plasmid containing cDNA encoding humanIL-1 by the colony hybridization assay as mentioned above.

Five clones having cDNA which could strongly be hybridized with thelabelled probe were selected from about 20,000 clones. Furthermore, 2clones harboring the recombinant plasmids containing cDNA of 2 kbp ormore in size were selected, and they were subjected to the mRNAhybridization translation assay. The plasmid DNA was extracted from eachof the selected clones and fixed to nitrocellulose filters after heatdenaturation. The poly(A)mRNA fraction containing human IL-1 mRNAobtained in the above (1) was added to the filters and incubated at 50°C. for 5 hours to perform hybridization. The hybridized mRNA wasrecovered and injected into the oocytes of Xenopus laevis to determinewhether the recovered mRNA was IL-1 mRNA. By this test, it was confirmedthat each of these 2 clones contained cDNA which could strongly behybridized with human IL-1 mRNA.

Out of these 2 clones, one clone harboring a recombinant plasmidcontaining cDNA of about 2.1 kbp in size was selected for sequencing thecloned cDNA (designated plasmid No. pHL4; clone No. X1776/pHL4).

(7) Determination of the nucleotide sequence of the cloned cDNA

The transformant (X1776/pHL4) selected in the above (6) was cultivatedin L broth supplemented with diaminopimelic acid and thymidine. Thecells were treated in accordance with the method of Wilkie et al.[Nucleic Acids Res., 7, 859 (1979)] to obtain a plasmid DNA. The plasmidDNA was cleaved with restriction endonuclease PstI, and purified toobtain a cloned cDNA insert. The cloned cDNA was further cleaved with asingle or two kinds of the following restriction endonucleases, SacI,RsaI, HindIII, HincII, Fnu4HI, HinfI, BalI and EcoRI. The resulting cDNAfragments being about 150-700 bp in size were isolated and used fordetermination of their nucleotide sequences.

The determination of nucleotide sequences were done by the dideoxymethod according to the text of "M13 cloning and sequencing handbood(Amersham International plc)" by using M13 sequencing kit (AmershamInternational plc), and M13mp18 and M13mp19 (product of P-LBiochemicals) as a cloning vector.

Table 5 shows the nucleotide sequence encoding human IL-1 and the aminoacid sequence deduced from the nucleotide sequence, wherein the codon ofbase No. 1 to base No. 3 is an initiation codon ATG and the codon ofbase No. 814 to base No. 816 is a stop codon TAG.

The nucleotide coding for human IL-1 encodes its precursor polypeptideconsisting of 271 amino acids (amino acid No. 1-271 in Table 5). Maturehuman IL-1 polypeptide is a polypeptide corresponding to the 159 aminoacids from the C-terminus of its precursor, which is coded in thenucleotide sequence from base No. 337 to base No. 813 in the Table 5.

Example 2 Cloning and sequencing of cDNA encoding rabbit IL-1

(1) Preparation of rabbit IL-1 mRNA from rabbit alveolar macrophagecells

Rabbits weighing about 2.5-3.0 kg were intravenously injected withkilled dried cells of Propionibacterium acnes at a dose of 100 mg perrabbit, and sacrificed 8 days later. The lungs were repeatedly washedwith phosphate buffered saline through a tube inserted into the tracheaof the animals, and alveolar macrophages were collected. The alveolarmacrophages were suspended in RPMI-1640 medium containing 10% fetalbovine serum, and seeded in Petri dishes (8 cm in diameter) at a celldensity of 1×10⁷ cells/dish. They were pre-incubated at 37° C. in afully humidified atmosphere containing 5% carbon dioxide. After 1 hourcultivation, LPS, TPA (phorbol-12-myristate-13acetate) and cycloheximidewere added to the dishes so that their final concentrations became 10micrograms/ml, 10 ng/ml and 1 microgram/ml, respectively. Thecultivation was further continued for 4 hours. The culture medium wasremoved by suction, and the macrophages adhered to the dishes were lysedand homogenized in a 6M guanidinium thiocyanate solution containing 0.5%sodium N-lauroyl sarcosinate, 5 mM sodium citrate and 0.1M2-mercaptoethanol. The homogenate was loaded on a cushion of a 5.7Mcesium chloride solution containing 0.1M EDTA, and centrifuged for 20hours at 26,500 rpm using an ultracentrifuge (RPS27-2 rotor, HitachiKoki) to obtain a total RNA fraction as pellets. The pellets weredissolved in a small amount of a 7M urea solution containing 0.35M NaCl,20 mM Tris-HCl (pH 7.4) and 20 mM EDTA, and recovered by precipitationfrom ethanol. The total RNA fraction was dissolved in 2 ml of the TEsolution, and the solution was heated at 65° C. for 5 minutes. A NaClsolution was added to a final concentration of 0.5M, and the solutionwas applied onto a column of ligo(dT)-cellulose previously equilibratedwith the TE solution containing 0.5M NaCl. Poly(A)mRNA was eluted fromthe column with the TE solution. The poly(A)-mRNA was obtained in anyield of about 300 micrograms from 1.3×10⁹ cells of rabbit alveolarmacrophages.

The resulting poly(A)mRNA was subjected to agarose gel electrophoresis(gel concentration 1%, in the presence of 6M urea at pH 4) according tothe method of Lehrach et al. [Biochemistry, 16, 4743 (1977)], and thepoly(A)mRNA fraction containing rabbit IL-1 mRNA was recovered from thegel fraction corresponding to about 2.6-3.7 kb in size by the method ofGray et al. [Nature, 295, 503 (1982)] (hereinafter referred to as"enriched poly(A)mRNA"). About 34 micrograms of the enriched poly(A)mRNAwere obtained from 200 micrograms of the poly(A)mRNA.

The enriched poly(A)mRNA was dissolved in distilled water at aconcentration of 0.2 microgram per microliter, and the solution wasinjected into the oocytes of Xenopus laevis at a dose of about 50 nl peroocyte, and the ten oocytes were incubated in 100 microliters of theBarth's medium [J. B. Gurdon, J. Embryol. Exp. Morphol. 20, 401 (1968)]at 22° C. for 24 hours. The oocytes were homogenized and centifuged. Thesupernatant was subjected to assay of IL-1 activity.

Each of 100-fold and 400-fold dilutions of the supernatant prepared asabove showed ³ H-thymidine incorporation of 7,979 cpm and 2,187 cpm intothe mouse thymocyte, respectively. It indicates that the enrichedpoly(A)mRNA preparation contains IL-1 mRNA.

(2) Synthesis of cDNA

Complementary DNA was synthesized according to the method of Gubler andHoffman [Gene, 25, 263 (1983)] using the enriched poly(A)mRNA obtainedin the above (1) as a template.

Four micrograms of the enriched poly(A)mRNA were dissolved in 50microliters of 50 mM Tris-HCl (pH 8.3) buffer containing 10 mM MgCl₂, 10mM dithiothreitol, 4 mM sodium pyrophosphate, 1.25 mM dGTP, 1.25 mMdATP, 1.25 mM dTTP, 0.5 mM dCTP, 170 nM alpha-³² P-dCTP (specificradioactivity, 750 Ci/mmole), 5 micrograms of cligo(dT)₁₂₋₁₈ and 120units of reverse transcriptase derived from AMV and incubated at 42° C.for 60 minutes. The reaction was stopped by adding EDTA. The reactionmixture was extracted with phenol/chloroform (1:1), and ammonium acetatewas added to the aqueous phase to a final concentration of 2.5M. Theresulting sscDNA-mRNA hybrid was recovered from the aqueous phase byprecipitation from ethanol. The sscDNA-mRNA hybrid precipitate wasdissolved in 100 microliters of 20 mM Tris-HCl (pH 7.5) buffercontaining 5 mM MgCl₂, 10 mM (NH₄)₂ SO₄, 100 mM KCl, 0.15 mMbeta-nicotinamide-adenine dinucleotide, 5 micrograms of bovine serumalbumin, 0.04 mM of each of four deoxyribonucleotide triphosphates,dGTP, dATP, dTTP, and dCTP, 12.5 units of E. coli ribonuclease H and 50units of E. coli DNA polymerase I. The reaction mixture was incubated at12° C. for 60 minutes, and further incubated at 22° C. for 60 minutes tosynthesize a dscDNA. The reaction was stopped by adding EDTA. The dscDNAwas extracted with phenol/chloroform and recovered by precipitation fromethanol as mentioned above.

(3) Preparation of cligo(dC)-tailed cDNA

The dscDNA obtained as above was dissolved in 100 microliters of 100 mMsodium cacodylate (pH 7.2) buffer containing 2 mM CoCl₂, 0.2 mMdithiothreitol, 0.1 mM alpha-³² P-dCTP (specific radioactivity, 1Ci/mmole) and 10 units of terminal deoxynucleotidyl transferase, andincubated at 37° C. for 30 minutes to permit the addition of oligo(dC)tails to the 3'-termini of dscDNA.

The reaction was stopped by adding EDTA. The oligo(dC)-tailed dscDNA wasextracted with phenol/chloroform, and recovered by precipitation fromethanol. The oligo(dC)-tailed dscDNA was dissolved in 10 mM Tris-HCl (pH7.4) buffer containing 1 mM EDTA and 100 mM NaCl so that it contained 2micrograms of the oligo(dC)-tailed dscDNA per ml.

(4) Construction of recombinant plasmids

Oligo(dG)-tailed pBR322 DNA (Product of Bethesda Res. Labs. Inc., USA)and the oligo(dC)-tailed dscDNA obtained in the above (3) were dissolvedin 10 mM Tris-HCl (pH 7.4) buffer containing 1 mM EDTA and 100 mM NaClso that they were contained in 1.5 micrograms and 0.09 microgram,respectively, in a total volume of 1.5 ml.

The mixture was incubated sequentially at 65° C. for 10 minutes, at 57°C. for 2 hours and at 45° C. for 2 hours to perform annealing and toconstruct recombinant plasmids.

(5) Selection of transformants

E. coli X1776 strain was transformed with the recombinant plasmidsobtained as above.

Specifically, E. coli X1776 was cultivated at 37° C. in 20 ml of L broth(composition: 10 g tryptone, 5 g yeast extract, 5 g NaCl and 1 g glucoseper liter; pH 7.2) supplemented with 100 micrograms/ml of diaminopimelicacid and 40 micrograms/ml of thymidine until the turbidity at 600 nmreached 0.5. The cells were collected by centrifugation at 4° C., andwashed with 10 ml of 10 mM Tris-HCl (pH 7.3) buffer containing 50 mMCaCl₂. The cells were resuspended in 2 ml of the same buffer as usedabove, and allowed to stand at 0° C. for 5 minutes. To 0.2 ml of thecell suspension was added 0.1 ml of the recombinant plasmid solutionobtained as above. The mixture was allowed to stand at 0° C. for 15minutes and then maintained at 42° C. for 2 minutes. Then, 0.5 ml of thesupplemented L broth as used above was added, and cultivation wascarried out with shaking for 1 hour. An aliquot of the culture wastaken, spread on the supplemented L broth agar plate containing 15micrograms/ml of tetracycline, and cultivated at 37° C. for about 12hours. A cDNA library was prepared by selecting transformants resistantto tetracycline.

(6) Cloning

In order to screen the cDNA library for transformants which had aplasmid containing cDNA encoding rabbit IL-1, colony hybridization assaywas conducted by the method of Hanahan and Meselson [Gene, 10, 63(1980)] using a ³² P-labelled induction-plus and induction-minus cDNAprobe. Each of induction-plus and induction-minus ³² P-labelled sscDNAprobes was synthesized respectively by the method described in the above(2) using as a template the enriched poly(A)mRNA fraction which wasfractionated by the acid-urea agarose gel electrophoresis from each ofthe alveolar macrophages induced with LPS, TPA and cycloheximide by themethod described in the above (1) and of the non-induced alveolarmacrophages. By this test, there were selected colonies of transformantsharboring the recombinant plasmids which strongly hybridized with theinduction-plus probe but did not hybridize with the induction-minusprobe. From about 5,000 colonies, 648 colonies were selected.

Ten groups of the selected colonies (including 10 colonies in eachgroup) were then subjected to mRNA hybridization-translation assay bythe method described in T. Maniatis et al. (ed) "Molecular Cloning", 329(1980), Cold Spring Harbor Lab. The plasmid DNAs were extracted from theeach group, and linealized by digestion with restriction endonucleaseEcoRI, and fixed to nitrocellulose filters after heat denaturation. Thepoly(A)mRNA fraction containing rabbit IL-1 mRNA obtained in the above(1) was added to the filter and incubated at 50° C. for 5 hours toperform hybridization. The hybridized mRNA was recovered and injectedinto the oocytes of Xenopus laevis to determine whether the recoveredmRNA was rabbit IL-1 mRNA. As a result of this test, it was found thatone group (designated H-1603) had plasmids containing cDNAs whichstrongly hybridized with the rabbit IL-1 mRNA (see FIG. 1-A). Each cloneof the group H-1603 was further subjected to the mRNAhybridization-translation assay as above, and it was found that oneclone was strongly hybridized with rabbit IL-1 mRNA (see FIG. 1-B). Thisselected clone was designated pRL15. The cDNA insert was cut out fromthe recombinant plasmid pRL15 by digestion with restriction endoncleasePstI, and it was labelled with ³² P. By using this ³² P-labelled cDNA asa probe, 648 colonies selected as above were screened by colonyhybridization assay again. As a result of this test, 8 clones werehybridized with the ³² P-labelled cDNA probe.

(7) Determination of the nucleotide sequence of the cloned cDNA

The recombinant plasmid pRL15 was cleaved with restriction endoncleasePstI, and purified to obtain a cloned cDNA insert. The cloned cDNA wasfurther cleaved with a single or two kinds of the following restrictionendonucleases, HaeIII, HincII, AccI, PvuII, AvaII and RsaI. Theresulting cDNA fragments being about 100-600 bp in size were used fornucleotide sequence determination.

The determination of nucleotide sequences were done by the dideoxymethod accoding to the text of "M13 cloning and sequencing handbook(Amersham International plc)", by using a M13 sequencing kit (AmershamInternational plc), and M13mp18 and M13mp19 (product of P-LBiochemicals) as a cloning vector.

Table 7 shows the nucleotide sequence and the amino acid sequencededuced from the nucleotide sequence encoding rabbit IL-1.

The codon of base No. 1 to base No. 3 is an initiation codon ATG, andthe codon of base No. 802 to base No. 804 is a stop codon TAA. The DNAcoding for rabbit IL-1 encodes its precursor polypeptide consisting of267 amino acid residues (amino acid No. 1-267 in Table 7). Mature rabbitIL-1 polypeptide is a polypeptide corresponding to the 155 amino acidsfrom the C-terminus of its precursor; which is coded in the nucleotidesequence from base No. 337 to base No. 801 in the Table 7.

Homology between the nucleotide sequences coding for human and rabbitIL-1 precursor polypeptides, and homology between the deduced amino acidsequences of human and rabbit IL-1 precursor polypeptides are shown inTable 8-1 and 8-2, respectively. Homologies in the nucleotide sequencesand the amino acid sequences are about 79% and 64%, respectively.

Example 3 Cloning and sequencing of human genomic IL-1 gene

Human genomic IL-1 gene was screened from the human genomic library,HaeIII-AluI digested human DNA fragments inserted into bacteriophageCharon 4A, which was supplied by Dr. T. Maniatis, [Harvard University,Department of Biochemistry and Molecular Biology, USA; Lawn, R. M. etal., Cell 15, 1157 (1978)] by the plaque hybridization method of Bentonand Davis [Science, 196, 180 (1977)] using as a probe the ³² P-labelledDNA fragmant as prepared below. From the recombinant plasmid pHL4obtained in Example 1-(6), the DNA fragment of about 847 bp in sizecorresponding to the downstream from the base No. 5 in Table 5, was cutout by double digestion with restriction endonucleases BalI and HincII,and labelled with ³² P by nick-translation method.

As a result, six phage clones containing the nucleotide sequence thatstrongly hybridized with the probe were isolated from the library ofabout 600,000 phage plaques. These clones were further screened for theclones which covered all the exons of human IL-1 gene by hybridizationmethod using the ³² P-labelled DNA fragment (about 1.1 kbp in size) cutout with restriction endonuclease HincII and HindIII from the cDNAinsert of recombinant plasmid pHL4 obtained in Example 1-(6) as a probe.The above HincII-HindIII DNA fragment contains most of the 3'-noncodingregion of human IL-1 cDNA. Out of clones selected as above, four cloneswere strongly hybridized with this probe. The recombinant phage DNA wasisolated from each clone and characterized by restriction endonucleasemapping. One clone (λHL#4) was selected for nucleotide sequencing.

FIG. 2 shows the structure of human IL-1 chromosomal gene includingexons and introns. Table 6 shows the nucleotide sequence in the regionsencoding human IL-1 precursor, and the amino acid sequence correspondingto human IL-1 precursor. In this table, the intron regions were shown bydotted lines.

The nucleotide sequence encoding human IL-1 obtained above agreescompletely with the nucleotide sequence of the cloned cDNA encodinghuman IL-1 as mentioned in Example 1.

Example 4 Production of human IL-1 polypeptide

(1) Construction of human IL-1 producing transformant

An expression plasmid designated for producing human IL-1 wasconstructed by using a trp promoter as illustrated in FIG. 3-5.

The cloned cDNA encoding human IL-1 was isolated from the recombinantplasmid pHL4 as mentioned in Example 1(6). Twenty micrograms of the cDNAwere dissolved in 100 microliters of 10 mM Tris-HCl (pH 7.5) buffercontaining 50 mM NaCl, 6 mM MgCl₂ and 6 mM 2-mercaptoethanol, and it wasadded with 240 units of restriction endonuclease HindIII, and thenincubated at 37° C. for 60 minutes. Then, 100 microliters of 0.2M NaCland 100 units of restriction endonuclease ScaI were added, and furtherincubated at 37° C. for 60 minutes. At the end of incubation, NaCl wasadded into the reaction mixture to give a final concentration of 0.3M,and the resulting DNA fragments were recovered by precipitation with 2volumes of ethanol. About 1.6 kbp DNA fragment containing a codingregion for human IL-1 was isolated by 5% polyacrylamide gelelectrophoresis, in an yield of about 5 micrograms.

This DNA fragment was ligated with a chemically synthesizedoligodeoxyribonucleotide adapter by T4 ligase.

The synthetic adaptor is represented by the following formula. ##STR1##

The resulting DNA fragment is referred to as the HIL-adaptor fragment.

The trp promoter region was cut out from a trp promoter vector pDR720[Russell, D. R., et al., Gene, 20, 231 (1982); product of P-LBiochemicals] by double digestion with restriction indonucleases EcoRIand HpaI, and a 35 bp DNA fragment containing the trp promoter regionwas isolated. A synthetic adaptor which was represented by the followingformula, was ligated to the blunt-end of the 35 bp DNA fragment by usingT4 ligase. The resulting DNA fragment is, hereinafter, referred to asthe "trp promoter fragment". ##STR2##

Separately, 20 micrograms of plasmid pBR322 was dissolved in 100microliters of 10 mM Tris-HCl (pH 7.5) buffer containing 50 mM NaCl, 6mM MgCl₂, 6 mM 2-mercaptoethanol and 180 units of restrictionendonuclease HindIII, and incubated at 37° C. for 60 minutes. Theresulting DNA was recovered by extraction with phenol/chloroform andprecipitation from ethanol. And this DNA fragment was dissolved in 20microliters of the TE solution. To ten microliters of the solution, 40microliters of 62.5 mM Tris-HCl (pH 7.2) buffer containing 12.5 mMMgCl₂, 0.125 mM dithiothreitol, 0.25 mM dGTP, 0.25 mM dATP, 0.25 mMdTTP, 0.25 mM dCTP, 2.5 micrograms of bovine serum albumin and 2.6 unitsof E. coli DNA polymerase I (large fragment), was added, and incubatedat 20° C. for 60 minutes. The resulting DNA fragment was recovered byextraction with phenol/chloroform and precipitation from ethanol. TheDNA fragment was dissolved in 20 microliters of the TE solution. Theabove procedure gave a linealized double stranded DNA fragment which wasdigested with restriction endonuclease HindIII and then repaired theboth ends to blunt-ends. Then, the DNA fragment was cut with restrictionendonuclease EcoRI into two fragments. A larger DNA fragment (about 4.3kbp) containing the ampicillin resistance gene was isolated (hereinafterreferred to as the "pBR322-Amp^(r) fragment").

The previously prepared HIL-adaptor fragment was ligated with the trppromoter fragment by T4 ligase at the ClaI cohesive termini of these twofragments. The resulting DNA fragment which had an EcoRI cohesive endand a blunt end, was ligated with the pBR322-Amp^(r) fragment by T4ligase to construct an expression plasmid (pHLP101) for production ofhuman IL-1.

The resulting expression plasmid pHLP101 was introduced into E. coliHB101 by the following method.

E. coli HB101 was inoculated in 5 ml of the L broth and cultivatedovernight at 37° C. One milliliter of the resulting culture wasinoculated in 100 ml of L broth, and further cultivated at 37° C. untilthe turbidity at 650 nm of the culture reached 0.6. After standing for30 minutes in ice water, the cells were collected by centrifugation andsuspended in 50 ml of 50 mM CaCl₂, followed by standing at 0° C. for 60minutes. The cells were then collected by centrifugation and againsuspended in 10 ml of 50 mM CaCl₂ containing 20% glycerol.

The expression plasmid pHLP101 was mixed with the calcium treated E.coli HB101 as above, and incubated in ice water for 20 minutes, then at42° C. for 1 minute and further at room temperature for 10 minutes, andLB broth was added. The mixture was shaken at 37° C. for 60 minutes. Analiquot of the resulting cell suspension was seeded on LB agar platescontaining 25 micrograms/ml of ampicillin, and cultivated overnight at37° C. Then, ampicillin-resistant colonies were selected to obtaintransformants. One of the transformants was named HB101/pHLP101 and itwas used for production of human IL-1.

(2) Production of human IL-1 polypeptide

The transformant (HB101/pHLP101) was cultivated at 37° C. overnight inthe LB broth. One-tenth milliliter of the culture was inoculated in 10ml of the modified M9 medium (composition: 1.5% Na₂ HPO₄.12H₂, 0.3% KH₂PO₄, 0.05% NaCl, 0.1% NH₄ Cl, 2 mg/liter vitamin B₁, 0.5% casamino acid,2 mM MgSO₄, 0.1 mM CaCl₂, and 0.5% glucose) and cultivated at 37° C. for1 hour. Then, 3-indoleacrylic acid was added to a final concentration of20 micrograms/ml, and the cultivation was continued further for 24hours. Then, the cells were collected by centrifugation. The cells weresuspended in 1 ml of 50 mM Tris-HCl (pH8.0) buffer containing 0.1%lysozyme and 30 mM NaCl and allowed to stand at 0° C. for 30 minutes.Further, freezing on a dry ice/ethanol bath and thawing at 37° C. wererepeated 6-times. And, the cell debris was removed by centrifugation togive a clarified lysate.

The IL-1 activity of the lysate was determined according to the methodas mentioned in Example 1-(1).

A 16-fold diluted solution of the lysate showed ³ H-thymidineincorporation of 8,103 cpm, which was corresponded to about 3-times ormore higher incorporation than control level. It indicates that thelysate prepared as above contains human IL-1 polypeptide.

The SDS-polyacrylamide gel electrophoretic pattern of the lysate wasshown in FIG. 6 by comparison with that of mature human IL-1 polypeptideobtained in Example 5-(2). The product produced by E. coli HB101/pHLP101is expected to be a polypeptide consisting of 209 amino acid residuescorresponding to the amino acid No. 63 to the amino acid No. 271 inTable 5. The expected molecular weight should be theoretically 23,642 ifN-terminal methionine was removed. As shown in FIG. 6, however, IL-1activity was detected in three fractions. The major peak having IL-1activity was detected at the position corresponding to 18 kilodaltons(KD). 18 KD polypeptide might be mature human IL-1 corresponding to theregion from the amino acid No. 113 to the amino acid No. 271 in Table 5.

This result indicates that the product might be gradually processed byan enzyme(s) in E. coli into polypeptides including mature human IL-1polypeptide.

Example 5 Production of mature human IL-1 polypeptide

(1) Construction of mature human IL-1 producing transformant

An expression plasmid (pHLP383) for producing mature human IL-1polypeptide consisting of 159 amino acids corresponding to the aminoacids from No. 113 to 271 shown in Table 5, was constructed asillustrated in FIG. 7.

The cloned cDNA encoding mature human IL-1 was isolated by digestionwith restriction endonuclease PstI from the recombinant plasmid pHL4 asmentioned in Example 1-(6). The cDNA was further digested withrestriction endonuclease AluI to obtain a DNA fragment being about 533bp in size corresponding to the DNA downstream from the base No. 351 inTable 5. Furthermore, the DNA fragment was digested with restrictionendonuclease BstNI to isolate the DNA fragment corresponding to the baseNo. 351 to 808 in Table 5. The resulting DNA fragment was sequentiallyligated by T4 ligase with chemically synthesizedoligodeoxyribonucleotide adaptors represented by the following formulae.##STR3##

The DNA fragment having the initiation codon ATG to the 5' end of theDNA fragment encoding mature human IL-1 polypeptide consisting of 159amino acids as mentioned above and the double stop codons TGATGA to the3' end of it was constructed (hereinafter, referred to as "HIL-1fragment".

About 380 bp DNA fragment containing the trp promoter region was cut outfrom a plasmid pCT-1 [Ikehara, M., et al., Proc. Natl. Acad. Sci. USA,81, 5956 (1984)] by double digestion with restriction endonucleases HpaIand AatII and isolated. The DNA fragment was ligated by T4 ligase with achemically synthesized oligodeoxyribonucleotide adaptor represented bythe following formula. ##STR4##

The resulting DNA fragment was ligated with the previously preparedHIL-1 fragment by T4 ligase (hereinafter referred to as the"promoter-HIL-1 fragment").

Separately, plasmid pBR322 was digested with restriction endonucleasesAvaI and PvuII, and the resulting larger DNA fragment (about 3.7 kbp insize) was isolated by 0.7% agarose gel electrophoresis. After filling-inits cohesive ends to blunt-ends with E. coli DNA polymerase I (largefragment) and four kinds of deoxyribonucleotide triphosphates, both endswere ligated by T4 ligase to construct a new plasmid (designated pBRS6).

The plasmid pBRS6 was cleaved with restriction endonuclease AatII andHindIII into two fragments, and a larger DNA fragment (about 3.6 kbp)was isolated. Then, this fragment was ligated by T4 ligase with thepromoter-HIL-1 fragment previously prepared in order to construct anexpression plasmid pHLP383.

The resulting expression plasmid pHLP383 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of mature human IL-1 was named HB101/pHLP383.

(2) Production of mature human IL-1 polypeptide

The transformant (HB101/pHLP383) was cultivated overnight in the LBbroth. Ten milliliters of the culture were inoculated in 1 liter of themodified M9 medium and cultivated at 37° C. for 1 hour. Then,3-indoleacrylic acid was added to a final concentration of 20micrograms/ml, and the cultivation was continued further for 24 hours.Then, the cells were collected by centrifugation. The cells weresuspended in 100 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 0.1%lysozyme and 30 mM NaCl and allowed to stand at 0° C. for 30 minutes.Then, the suspension was freezed in a dry ice/ethanol bath and thawed at37° C. After this freezing-thawing procedure was repeated 6-times, 2 mlof a 10% polyethyleneimine solution was added to the cell suspension andit was allowed to stand. The cell debris was removed by centrifugationto give a clarified extract.

The extract was mixed with an equal volume of saturated ammonium sulfatesolution. After standing, a precipitate was collected by centrifugation.The precipitate was dissolved in about 100 ml of 20 mM Tris-HCl (pH 8.0)buffer, and dialyzed against the same buffer. The dialysate was appliedonto the column of DEAE-Sepharose CL-6B which was previouslyequilibrated with the same buffer. The column was washed with the samebuffer, and eluted with a linear gradient of NaCl from 0 to 0.5M. Thefractions having IL-1 activity were collected and pooled. Then, it wasconcentrated by ultrafiltration and subjected to gel filtration using aSephacryl S-200 column. The fractions having IL-1 activity werecollected and pooled. The purified preparation was obtained by repeatingthe above procedures of DEAE-Sepharose CL-6B column chromatography andSephacryl S-200 gel filtration.

Finally, about 15 mg of the purified human IL-1 polypeptide wereobtained from the cell extract prepared from 1 liter culture.

By SDS-polyacrylamide gel electrophoretic analysis of the purifiedpreparation, only a single protein having IL-1 activity was detected andany impurities were not detected. The chemical and physiochemicalproperties of the purified mature human IL-1 polypeptide were as shownin Table (a) hereinbefore.

The IL-1 activity of the purified human IL-1 polypeptide solution(approx. 40 micrograms/ml) was determined, and its results weresummarized in the following table.

                  TABLE (d)                                                       ______________________________________                                        IL-1 activity                                                                 dilution of sample .sup.3 H-thymidine                                         (fold)             incorporated (cpm)                                         ______________________________________                                           100,000         32,746 cpm                                                  1,000,000         14,498                                                      10,000,000         4,206                                                     100,000,000         2,582                                                     control             3,210                                                     ______________________________________                                    

The purified mature IL-1 polypeptide was soluble in phosphate bufferedsaline at about 1%.

(3) Antitumor effect of mature human IL-1 polypeptide

The purified mature human IL-1 polypeptide obtained above wasinvestigated on antitumor effect on B-16 melanoma and Meth A sarcomatransplanted into mice.

The antitumor effect on mice bearing B-16 melanoma was evaluated by thefollowing method.

C57BL/6 female mice (6 week old) were intradermally transplanted with0.1 ml of 20% brei. On the 7th day after the tumor transplantation,mature human IL-1 polypeptide obtained in Example 5-(2) was administeredinto the tumor mass, intramuscularly or intravenously. Endotoxin contentof the mature human IL-1 polypeptide preparation used was less than 0.08ng per mg protein.

As a result, the growth of B-16 melanoma transplanted was significantlyregressed by consecutive administration for 7 days at a dose of 3micrograms/mouse/day into the tumor mass, showing the growth inhibitionrate of 79%. And by increasing its dose upto 10-fold, the B-16. melanomatransplanted was completely regressed in 6 cut of 7 mice. In the casesof intravenous and intramuscular injection at a dose of 30micrograms/mouse/day for 7 days from the 7th day after the tumortransplantation, the growth of B-16 melanoma transplanted wassignificantly suppressed at inhibition rates of 45% and 68%,respectively.

Antitumor effect on mice bearing Meth A sarcoma was evaluated by thefollowing method.

BALB/c female mice (8 week old) were intradermally transplanted with2×10⁵ Meth A sarcoma cells. On the 7th day after the tumortransplantation, indomethacin was previously administered orally at adose of 2 mg/kg. At 30 minutes later, mature human IL-1 polypeptide wasintramuscularly injected once at a dose of 30 micrograms/mouse, and thenindomethacin was administered orally twice at the same dose at 6 and 24hours after IL-1 injection.

As a result, Meth A sarcoma transplanted was significantly inhibited togrow at the growth inhibition rates of 96% and 62% by treatment ofmature human IL-1 polypeptide with and without indomethacin,respectively. The rates of complete regression was 3/7 and 2/7,respectively.

(4) Antiinfectious effect of mature human IL-1 polypeptide

Std-ddY male mice weighing about 20 g were intraperitoneally infectedwith 1.5×10¹ Klebsiella pneumoniae P-5709 cells per mouse. Mature humanIL-1 polypeptide was intramuscularly (im) injected once daily atindicated doses at indicated days prior to the infection as shown inTable (e). Mortality was evaluated at 14 days after the infection.

Mature human IL-1 polypeptide showed prophylactic and therapeuticeffects on the infection as shown in the following table.

                  TABLE (e)                                                       ______________________________________                                        Prophylactic and therapeutic effects                                          of the experimental infection                                                 Medication       No. of mice   Percent                                        Route*    Dose       (Survival/Tested)                                                                           Survival                                   ______________________________________                                        im          3 μg/mouse                                                                          3/8           37.5                                       (-3 d, -1 d)                                                                              1 μg/mouse                                                                          0/8           0                                                    0.3 μg/mouse                                                                          0/8           0                                                    0.1        0/8           0                                          im          3 μg/mouse                                                                          8/8           100                                        (-1 d, 0 h)                                                                               1 μg/mouse                                                                          1/8           12.5                                                 0.3 μg/mouse                                                                          0/8           0                                                    0.1 μg/mouse                                                                          0/8           0                                          im          3 μg/mouse                                                                          8/8           100                                        (0 h, 24 h)                                                                               1 μg/mouse                                                                          5/8           62.5                                                 0.3        0/8           0                                                    0.1        0/8           0                                          Control infected 0/8           0                                              ______________________________________                                         [Note]: *)-3 d, -1 d, 0 h and 24 h mean the IL1 injection at 3 days befor     the infection, 1 day before, immediately after, and 24 hours after,           respectively.                                                            

Example 6 Production of a modified polypeptide of human IL-1 polypeptide

(1) Construction of human IL-1(157) producing transformant

An expression plasmid (pHLP384) for producing human IL-1 polypeptideconsisting of 157 amino acids corresponding to the amino acids from No.115 to 271 shown in Table 5, which was referred to as IL-1(157)polypeptide, was constructed according to the essentially same method asmentioned in Example 5-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [c]. ##STR5##

The resulting expression plasmid pHLP384 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(157) polypeptide was named HB101/pHLP384.

(2) Production of human IL-1(157) polypeptide

The transformant (HB101/pHLP384) was cultivated overnight in the LBbroth. Ten milliliters of the culture were inoculated in 1 liter of themodified M9 medium and cultivated at 37° C. for 1 hour. Then,3-indoleacrylic acid was added to a final concentration of 20micrograms/ml, and the cultivation was continued further for 24 hours.Then, the cells were collected by centrifugation. The cells weresuspended in 100 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 0.1%lysozyme and 30 mM NaCl and allowed to stand at 0° C. for 30 minutes.Then, the suspension was freezed in a dry ice/ethanol bath and thawed at37° C. After this freezing-thawing procedure was repeated 6-times, 2 mlof 10% polyethyleneimine solution was added to the cell suspension andit was allowed to stand. The cell debris was removed by centrifugationto give a clarified extract.

The extract was mixed with an equal volume of saturated ammonium sulfatesolution. After standing, a precipitate was collected by centrifugation.The precipitate was dissolved in about 100 ml of 20 mM Tris-HCl (pH 8.0)buffer, and dialyzed against the same buffer. The dialysate was appliedonto the column of DEAE-Sepharose CL-6B which was previouslyequilibrated with the same buffer. The column was washed with the samebuffer, and eluted with a linear gradient of NaCl from 0 to 0.5M. Thefractions having IL-1 activity were collected and pooled. Then, it wasconcentrated by ultrafiltration and subjected to gel filtration by usinga Sephacryl S-200 column. The fractions having IL-1 activity werecollected and pooled.

The resulting solution had IL-1 activity, showing ³ H-thymidineincorporation of 45,394 cpm when 1×10⁴ -fold sample dilution wasassayed.

Chemical and physicochemical properties of the human IL-1(157)polypeptide are as shown in Table (b) hereinbefore.

Example 7 Production of a modified polypeptide of human IL-1 polypeptide

(1) Construction of human IL-1 (149) producing transformant

An expression plasmid (pHLP385) for producing human IL-1 polypeptideconsisting of 149 amino acids corresponding to the amino acids from No.123 to 271 shown in Table 5, which was referred to as IL-1(149)polypeptide, was constructed as illustrated in FIG. 8.

The recombinant plasmid pHLP383 obtained in Example 5-(1) was digestedwith restriction endonucleases EcoRI and HindIII to isolate about 422 bpDNA fragment corresponding to the nucleotide sequence of the downstreamregion from the base No. 398 in Table 5. The resulting DNA fragment wasligated by T4 ligase with a chemically synthesizedoligodeoxyribonucleotide adaptor represented by the following formula.##STR6##

Separately, the recombinant plasmid pHLP383 was cleaved with restrictionendonucleases ClaI and HindIII, and the resulting larger DNA fragmentcontaining a part of trp promoter region, an ampicillin resistance geneand a tetracycline resistance gene was isolated.

To this DNA fragment, the previously prepared DNA fragment was ligatedby T4 ligase in order to construct an expression plasmid pHLP385 forproduction of the above polypeptide consisting of 149 amino acids.

The resulting expression plasmid pHLP385 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(149) polypeptide was named HB101/pHLP385.

(2) Production of human IL-1(149) polypeptide

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP385) was cultivated, and then the desired-polypeptide wasisolated from the cell extract.

The obtained polypeptide had IL-1 activity, showing ³ H-thymidineincorporation of 27,766 cpm when 1×10⁴ -fold sample dilution wasassayed.

Chemical and physicochemical properties are as shown in Table (b)hereinbefore.

Example 8 Production of a modified polypeptide of human IL-1 polypeptide

(1) Construction of human IL-1(144) producing transformant

An expression plasmid (pHLP386) for producing human IL-1 polypeptideconsisting of amino acids corresponding to the amino acids from No. 127or 128 to 271 shown in Table 5, which was referred to as IL-1(144)polypeptide, was constructed according to the method as mentioned inExample 7-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [g]. ##STR7##

The resulting expression plasmid pHLP386 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(144) polypeptide was named HB101/pHLP386.

(2) Production of human IL-1(144) polypeptide

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP386) was cultivated, and then the desired polypeptide wasisolated from the cell extract.

The obtained polypeptide had IL-1 activity, showing ³ H-thymidineincorporation of 15,092 cpm when 1×10³ -fold sample dilution wasassayed.

Chemical and physicochemical properties of human IL-1 polypeptide are asshown in Table (b) hereinbefore.

Example 9 Production of a modified polypeptide of human IL-1 polypeptide

(1) Construction of human IL-1(143) producing transformant

An expression plasmid (pHLP387) for producing human IL-1 polypeptideconsisting of 143 amino acids corresponding to the amino acids from No.129 to 271 shown in Table 5 which was referred to as IL-1(143)polypeptide, was constructed according to the method as mentioned inExample 7-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [g]. ##STR8##

The resulting expression plasmid pHLP387 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(143) polypeptide was named HB101/pHLP387.

(2) Production of human IL-1(143) polypeptide

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP387) was cultivated, and then the desired polypeptide wasisolated from the cell extract.

The obtained polypeptide had IL-1 activity, showing ³ H-thymidineincorporation of 12,700 cpm when 10-fold sample dilution was assayed.

Example 10 Production of a modified polypeptide of human IL-1polypeptide

(1) Construction of human IL-1(155-C) producing transformant

An expression plasmid (pHLP373) for producing human IL-1 polypeptideconsisting of 155 amino acids corresponding to the amino acids from No.113 to 267 shown in Table 5, was constructed as illustrated in FIG. 9.

The cloned cDNA encoding human IL-1 was isolated by digestion withrestriction endonuclease PstI from the recombinant plasmid pHL4 asmentioned in Example 1-(6). The cDNA was further digested withrestriction endonuclease AluI to obtain a DNA fragment being about 533bp in size corresponding to the DNA downstream from the base No. 351 inTable 5. Furthermore, the DNA fragment was digested with restrictionendonuclease Sau96I to isolate the DNA fragment corresponding to thebase No. 351 to 769 in Table 5. The resulting DNA fragment wassequentially ligated by T4 ligase with chemically synthesizedoligodeoxyribonucleotide adaptors represented by the following formulae.##STR9##

The DNA fragmant having the initiation codon ATG at the 5' end of theDNA fragment encoding human IL-1 polypeptide consisting of 155 aminoacids as mentioned above and the double stop codons TGATGA at the 3' endof it was constructed (hereinafter referred to as "HIL-155 fragment").

About 380 bp DNA fragment containing the trp promoter region was cut outfrom a plasmid pCT-1 by double digestion with restriction endonucleasesHpaI and AatII and was isolated. The DNA fragment was ligated by T4ligase with a chemically synthesized oligodeoxyribonucleotide adaptorrepresented by the following formula. ##STR10##

The resulting DNA fragment was ligated with the previously prepared theHIL-155 fragment by T4 ligase (hereinafter referred to as the"promoter-HIL-155 fragment").

Separately, plasmid pBR322 was digested with restriction endonucleasesAvaI and PvuII, and the resulting larger DNA fragment (about 3.7 kbp insize) was isolated by 0.7% agarose gel electrophoresis. After filling-inits cohesive ends to blunt-ends with E. coli DNA polymerase I (largefragment) and four kinds of deoxyribonucleotide triphosphates, both endswere ligated by T4 ligase to construct a plasmid (designated pBRS6).

The plasmid pBRS6 was cleaved with restriction endonucleases AatII andHindIII into two fragments, and a larger DNA fragment (about 3.6 kbp)was isolated. Then, this fragment was ligated by T4 ligase with thepromoter-HIL-155 fragment previously prepared in order to construct anexpression plasmid pHLP373.

The resulting expression plasmid pHLP373 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(155-C) polypeptide was named HB101/pHLP373.

(2) Production of human IL-1(155-C) polypeptide

The transformant (HB101/pHLP373) was cultivated overnight in the LBbroth. Ten milliliters of the culture were inoculated in 1 liter of themodified M9 medium and cultivated at 37° C. for 1 hour. Then,3-indoleacrylic acid was added to a final concentration of 20micrograms/ml, and the cultivation was continued further for 24 hours.Then, the cells were collected by centrifugation. The cells weresuspended in 100 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 0.1%lysozyme and 30 mM NaCl and allowed to stand at 0° C. for 30 minutes.Further, freezing on a dry ice/ethanol bath and thawing at 37° C. wererepeated 6-times. And, the cell debris was removed by centrifugation togive a clarified extract.

The cell extract had IL-1 activity, showing ³ H-thymidine incorporationof 27,109 cpm when 1×10⁵ -fold sample dilution was assayed.

Chemical and physicochemical properties of human IL-1(155-C) polypeptideare shown in Table (c) hereinbefore.

Example 11 Production of a modified polypeptide of human IL-1polypeptide

(1) Construction of human IL-1(152-C) producing transformant andproduction thereof

An expression plasmid (pHLP358) for producing human IL-1 polypeptideconsisting of 152 amino acids corresponding to the amino acids from No.113 to 264 shown in Table 5, which was referred to as IL-1(152-C)polypeptide, was constructed according to the method as mentioned inExample 10-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [j]. ##STR11##

The resulting recombinant plasmid pHLP358 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(152-C) polypeptide was named HB101/pHLP358.

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP358) was cultivated, and then the desired polypeptide wasisolated from the cell extract.

Example 12 Production of a modified polypeptide of human IL-1polypeptide

(1) Construction of human IL-1(154-C) producing transformant

An expression plasmid (pHLP363) for producing human IL-1 polypeptideconsisting of 154 amino acids corresponding to the amino acids from No.113 to 266 shown in Table 5, which was referred to as IL-1(154-C)polypeptide, was constructed according to the method as mentioned inExample 10-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [j] ##STR12##

The resulting expression plasmid pHLP363 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(154-C) polypeptide was named HB101/pHLP363.

(2) Production of human IL-1(154-C) polypeptide

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP363) was cultivated, and then the desired polypeptide wasisolated from the cell extract.

The cell lysate had IL-1 activity, showing ³ H-thymidine incorporationof 11,058 cpm when 1×10² -fold sample dilution was assayed. Molecularweight of the obtained polypeptide having IL-1 activity was estimatedabout 17,500 daltons by SDS-polyacrylamide gel electrophoresis.

Example 13 Production of a modified polypeptide of human IL-1polypeptide

(1) Construction of human IL-1(153-C) producing transformant

An expression plasmid (pHLP353) for producing human IL-1 polypeptideconsisting of 153 amino acids corresponding to the amino acids from No.113 to 265 shown in Table 5, which was referred to as IL-1(153-C)polypeptide, was constructed according to the method as mentioned inExample 10-(1), except for using a chemically synthesizedoligodeoxyribonucleotide adaptor as shown below instead of the syntheticadaptor [j]. ##STR13##

The resulting expression plasmid pHLP353 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(153-C) polypeptide was nambed HB101/pHLP353.

(2) Production of human IL-1(153-C) polypeptide

According to the method as mentioned in Example 6-(2), the transformant(HB101/pHLP353) was cultivated, and then the desired polypeptide wasisolated from the cell extract.

The cell lysate had IL-1 activity, showing ³ H-thymidine incorporationof 3,875 cpm when 10-fold sample dilution was assayed. Molecular weightof the obtained polypeptide having IL-1 activity was estimated about17,500 daltons by SDS-polyacrylamide gel electrophoresis.

Example 14 Production of a modified polypeptide of human IL-1polypeptide

(1) Construction of human IL-1(140-NC) producing transformant

An expression plasmid (pHLP376) for producing human IL-1 polypeptideconsisting of 140 amino acids corresponding to the amino acids from No.128 to 267 shown in Table 5, was constructed.

An expression plasmid pHLP386 obtained in Example 8 was digested withrestriction endonuclease EcoRI and HindIII, and a resulting larger DNAfragment was isolated. Separately, an expression plasmid pHLP373obtained in Example 10-(1) was digested with restriction endonucleasesEcoRI and HindIII, and a resulting smaller DNA fragment was isolated.Then, the both DNA fragment as above were ligated by T4 ligase in orderto construct an expression plasmid for producing the above polypeptideconsisting of 140 amino acids (designated pHLP376).

The resulting recombinant plasmid pHLP376 was introduced into E. coliHB101 by the method described in Example 4-(1). The transformant forproduction of human IL-1(140-NC) was named HB101/pHLP376.

(2) Production of human IL-1(140-NC) polypeptide

The transformant (HB101/pHLP376) was cultivated overnight in the LBbroth. Ten milliliters of the culture were inoculated in 1 liter of themodified M9 medium and cultivated at 37° C. for 1 hour. Then,3-indoleacrylic acid was added to a final concentration of 20micrograms/ml, and the cultivation was continued further for 24 hours.Then, the cells were collected by centrifugation. The cells weresuspended in 100 ml of 50 mM Tris-HCl (pH 8.0) buffer containing 0.1%lysozyme and 30 mM NaCl and allowed to stand at 0° C. for 30 minutes.Further, freezing on a dry ice/ethanol bath and thawing at 37° C. wererepeated 6-times. And, the cell debris was removed by centrifugation togive a clarified extract.

The cell extract had IL-1 activity, showing ³ H-thymidine incorporationof 12,493 cpm when 1×10² -fold sample dilution was assayed. Molecularweight of the obtained polypeptide having IL-1 activity was estimatedabout 16,000 daltons by SDS-polyacrylamide gel electrophoresis.

Example 15 Production of mature rabbit IL-1 polypeptide

An expression plasmid for producing mature rabbit IL-1 was constructedby using a trp promoter as illustrated in FIG. 10.

The cloned cDNA insert containing a coding region for mature rabbit IL-1was isolated from the recombinant plasmid pRL15 obtained in Example2-(6) by digestion with restriction endonuclease PstI. The cDNA wasfurther digested with restriction endonucleases HincII and BglII toisolate a DNA fragment (about 509 bp in size). Then, the 509bp-DNAfragment was partially digested with restriction endonuclease DpnI andthe resulting DNA containing most of the coding region for mature rabbitIL-1 was isolated by 5% polyacrylamide gel electrophoresis.

This DNA fragment was ligated with chemically synthesizedoligodeoxyribonucleotide adaptors by T4 ligase.

The synthetic adaptors are represented by the following formulae.##STR14##

The resulting DNA fragment is referred to as the RIL-adaptor fragment.

Separately, about 380 bp DNA fragment containing the trp promoter regionwas cut out from a plasmid pCT-1 by double digestion with restrictionendonucleases HpaI and AatII, and it was isolated. The DNA fragment wasligated by T4 ligase with a chemically synthesizedoligodeoxyribonucleotide adaptor represented by the following formula.##STR15##

The resulting DNA fragment was sequentially ligated with the previouslyprepared the RIL-1-adaptor fragment and the AatII-HindIII 3.6 bp DNAfragment of plasmid pBRS6 obtained in Example 5-(1) by T4 ligase, inorder to construct an expression plasmid pRLP383.

By introducing the expression plasmid pRLP383 into E. coli HB101 inaccordance with the method described in Example 4-(1) and cultivatingthe transformant, mature rabbit IL-1 polypeptide consisting of 155 aminoacids corresponding to the amino acids from No. 113 to 267 shown inTable 7 will be able to be obtained.

                                      TABLE 1-1                                   __________________________________________________________________________    Ser                                                                              Ser                                                                              Pro                                                                              Phe                                                                              Ser                                                                              Phe                                                                              Leu                                                                              Ser                                                                              Asn                                                                              Val                                                                              Lys                                                                              Tyr                                                                              Asn                                                                              Phe                                                                              Met                                 Arg                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Tyr                                                                              Glu                                                                              Phe                                                                              Ile                                                                              Leu                                                                              Asn                                                                              Asp                                                                              Ala                                                                              Leu                                                                              Asn                                                                              Gln                                 Ser                                                                              Ile                                                                              Ile                                                                              Arg                                                                              Ala                                                                              Asn                                                                              Asp                                                                              Gln                                                                              Tyr                                                                              Leu                                                                              Thr                                                                              Ala                                                                              Ala                                                                              Ala                                                                              Leu                                 His                                                                              Asn                                                                              Leu                                                                              Asp                                                                              Glu                                                                              Ala                                                                              Val                                                                              Lys                                                                              Phe                                                                              Asp                                                                              Met                                                                              Gly                                                                              Ala                                                                              Tyr                                                                              Lys                                 Ser                                                                              Ser                                                                              Lys                                                                              Asp                                                                              Asp                                                                              Ala                                                                              Lys                                                                              Ile                                                                              Thr                                                                              Val                                                                              Ile                                                                              Leu                                                                              Arg                                                                              Ile                                                                              Ser                                 Lys                                                                              Thr                                                                              Gln                                                                              Leu                                                                              Tyr                                                                              Val                                                                              Thr                                                                              Ala                                                                              Gln                                                                              Asp                                                                              Glu                                                                              Asp                                                                              Gln                                                                              Pro                                                                              Val                                 Leu                                                                              Leu                                                                              Lys                                                                              Glu                                                                              Met                                                                              Pro                                                                              Glu                                                                              Ile                                                                              Pro                                                                              Lys                                                                              Thr                                                                              Ile                                                                              Thr                                                                              Gly                                                                              Ser                                 Glu                                                                              Thr                                                                              Asn                                                                              Leu                                                                              Leu                                                                              Phe                                                                              Phe                                                                              Trp                                                                              Glu                                                                              Thr                                                                              His                                                                              Gly                                                                              Thr                                                                              Lys                                                                              Asn                                 Tyr                                                                              Phe                                                                              Thr                                                                              Ser                                                                              Val                                                                              Ala                                                                              His                                                                              Pro                                                                              Asn                                                                              Leu                                                                              Phe                                                                              Ile                                                                              Ala                                                                              Thr                                                                              Lys                                 Gln                                                                              Asp                                                                              Tyr                                                                              Trp                                                                              Val                                                                              Cys                                                                              Leu                                                                              Ala                                                                              Gly                                                                              Gly                                                                              Pro                                                                              Pro                                                                              Ser                                                                              Ile                                                                              Thr                                 Asp                                                                              Phe                                                                              Gln                                                                              Ile                                                                              Leu                                                                              Glu                                                                              Asn                                                                              Gln                                                                              Ala                                                   [I-1]                                                                         __________________________________________________________________________

                                      TABLE 1-2                                   __________________________________________________________________________    (5')TCA                                                                            TCA CCT TTT AGC TTC CTG AGC AAT GTG AAA TAC AAC TTT                        ATG                                                                              AGG ATC ATC AAA TAC GAA TTC ATC CTG AAT GAC GCC CTC                        AAT                                                                              CAA AGT ATA ATT CGA GCC AAT GAT CAG TAC CTC ACG GCT                        GCT                                                                              GCA TTA CAT AAT CTG GAT GAA GCA GTG AAA TTT GAC ATG                        GGT                                                                              GCT TAT AAG TCA TCA AAG GAT GAT GCT AAA ATT ACC GTG                        ATT                                                                              CTA AGA ATC TCA AAA ACG CAA TTG TAT GTG ACG GCC CAA                        GAT                                                                              GAA GAC CAA CCA GTG CTG CTG AAG GAG ATG CCT GAG ATA                        CCC                                                                              AAA ACC ATC ACA GGT AGT GAG ACC AAC CTC CTC TTC TTC                        TGG                                                                              GAA ACG CAC GGC ACG AAG AAC TAT TTC ACA TCA GTT GCC                        CAT                                                                              CCA AAC TTG TTT ATT GCC ACA AAG CAA GAC TAC TGG GTG                        TGC                                                                              TTG GCA GGG GGG CCA CCC TCT ATC ACG GAC TTT CAG ATA                        CTG                                                                              GAA AAC CAG GCG(3')                                                      [II-1]                                                                        __________________________________________________________________________

                                      TABLE 2-1                                   __________________________________________________________________________    Met                                                                              Ala                                                                              Lys                                                                              Val                                                                              Pro                                                                              Asp                                                                              Met                                                                              Phe                                                                              Glu                                                                              Asp                                                                              leu                                                                              Lys                                                                              Asn                                                                              Cys                                                                              Tyr                                 Ser                                                                              Glu                                                                              Asn                                                                              Glu                                                                              Glu                                                                              Asp                                                                              Ser                                                                              Ser                                                                              Ser                                                                              Ile                                                                              Asp                                                                              His                                                                              Leu                                                                              Ser                                                                              Leu                                 Asn                                                                              Gln                                                                              Lys                                                                              Ser                                                                              Phe                                                                              Tyr                                                                              His                                                                              Val                                                                              Ser                                                                              Tyr                                                                              Gly                                                                              Pro                                                                              Leu                                                                              His                                                                              Glu                                 Gly                                                                              Cys                                                                              Met                                                                              Asp                                                                              Gln                                                                              Ser                                                                              Val                                                                              Ser                                                                              Leu                                                                              Ser                                                                              Ile                                                                              Ser                                                                              Glu                                                                              Thr                                                                              Ser                                 Lys                                                                              Thr                                                                              Ser                                                                              Lys                                                                              Leu                                                                              Thr                                                                              Phe                                                                              Lys                                                                              Glu                                                                              Ser                                                                              Met                                                                              Val                                                                              Val                                                                              Val                                                                              Ala                                 Thr                                                                              Asn                                                                              Gly                                                                              Lys                                                                              Val                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Arg                                                                              Arg                                                                              Leu                                                                              Ser                                                                              Leu                                                                              Ser                                                                              Gln                                 Ser                                                                              Ile                                                                              Thr                                                                              Asp                                                                              Asp                                                                              Asp                                                                              Leu                                                                              Glu                                                                              Ala                                                                              Ile                                                                              Ala                                                                              Asn                                                                              Asp                                                                              Ser                                                                              Glu                                 Glu                                                                              Glu                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Pro                                                                              Arg                                                         [I-2]                                                                         __________________________________________________________________________

                                      TABLE 2-2                                   __________________________________________________________________________    (5')ATG                                                                            GCC AAA GTT CCA GAC ATG TTT GAA GAC CTG AAG AAC TGT                        TAC                                                                              AGT GAA AAT GAA GAA GAC AGT TCC TCC ATT GAT CAT CTG                        TCT                                                                              CTG AAT CAG AAA TCC TTC TAT CAT GTA AGC TAT GGC CCA                        CTC                                                                              CAT GAA GGC TGC ATG GAT CAA TCT GTG TCT CTG AGT ATC                        TCT                                                                              GAA ACC TCT AAA ACA TCC AAG CTT ACC TTC AAG GAG AGC                        ATG                                                                              GTG GTA GTA GCA ACC AAC GGG AAG GTT CTG AAG AAG AGA                        CGG                                                                              TTG AGT TTA AGC CAA TCC ATC ACG GAT GAT GAC CTG GAG                        GCC                                                                              ATC GCC AAT GAC TCA GAG GAA GAA ATC ATC AAG CCT AGG(3')                  [II-2]                                                                        __________________________________________________________________________

                                      TABLE 3-1                                   __________________________________________________________________________    Ser                                                                              Val                                                                              Pro                                                                              Tyr                                                                              Thr                                                                              Phe                                                                              Gln                                                                              Arg                                                                              Asn                                                                              Met                                                                              Arg                                                                              Tyr                                                                              Lys                                                                              Tyr                                                                              Leu                                 Arg                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Gln                                                                              Glu                                                                              Phe                                                                              Thr                                                                              Leu                                                                              Asn                                                                              Asp                                                                              Ala                                                                              Leu                                                                              Asn                                                                              Gln                                 Ser                                                                              Leu                                                                              Val                                                                              Arg                                                                              Asp                                                                              Thr                                                                              Ser                                                                              Asp                                                                              Gln                                                                              Tyr                                                                              Leu                                                                              Arg                                                                              Ala                                                                              Ala                                                                              Pro                                 Leu                                                                              Gln                                                                              Asn                                                                              Leu                                                                              Gly                                                                              Asp                                                                              Ala                                                                              Val                                                                              Lys                                                                              Phe                                                                              Asp                                                                              Met                                                                              Gly                                                                              Val                                                                              Tyr                                 Met                                                                              Thr                                                                              Ser                                                                              Glu                                                                              Asp                                                                              Ser                                                                              Ile                                                                              Leu                                                                              Pro                                                                              Val                                                                              Thr                                                                              Leu                                                                              Arg                                                                              Ile                                                                              Ser                                 Gln                                                                              Thr                                                                              Pro                                                                              Leu                                                                              Phe                                                                              Val                                                                              Ser                                                                              Ala                                                                              Gln                                                                              Asn                                                                              Glu                                                                              Asp                                                                              Glu                                                                              Pro                                                                              Val                                 Leu                                                                              Leu                                                                              Lys                                                                              Glu                                                                              Met                                                                              Pro                                                                              Glu                                                                              Thr                                                                              Pro                                                                              Arg                                                                              Ile                                                                              Ile                                                                              Thr                                                                              Asp                                                                              Ser                                 Glu                                                                              Ser                                                                              Asp                                                                              Ile                                                                              Leu                                                                              Phe                                                                              Phe                                                                              Trp                                                                              Glu                                                                              Thr                                                                              Gln                                                                              Gly                                                                              Asn                                                                              Lys                                                                              Asn                                 Tyr                                                                              Phe                                                                              Lys                                                                              Ser                                                                              Ala                                                                              Ala                                                                              Asn                                                                              Pro                                                                              Gln                                                                              Leu                                                                              Phe                                                                              Ile                                                                              Ala                                                                              Thr                                                                              Lys                                 Pro                                                                              Glu                                                                              His                                                                              Leu                                                                              Val                                                                              His                                                                              Met                                                                              Ala                                                                              Arg                                                                              Gly                                                                              Leu                                                                              Pro                                                                              Ser                                                                              Met                                                                              Thr                                 Asp                                                                              Phe                                                                              Gln                                                                              Ile                                                                              Ser                                                               [A-1]                                                                         __________________________________________________________________________

                                      TABLE 3-2                                   __________________________________________________________________________    (5')TCA                                                                            GTT CCT TAC ACC TTC CAG AGA AAT ATG AGA TAC AAA TAC                        TTG                                                                              AGA ATC ATC AAA CAA GAG TTT ACC CTG AAT GAT GCT CTC                        AAT                                                                              CAA AGC CTA GTT CGT GAC ACC TCA GAT CAG TAC CTC AGG                        GCA                                                                              GCT CCA CTA CAA AAT CTG GGC GAT GCA GTG AAA TTT GAC                        ATG                                                                              GGG GTC TAC ATG ACA TCA GAG GAT TCT ATA CTT CCT GTA                        ACG                                                                              TTA AGA ATC TCA CAA ACT CCA CTG TTT GTG AGT GCC CAG                        AAT                                                                              GAA GAT GAA CCT GTG CTG CTA AAG GAA ATG CCT GAG ACA                        CCC                                                                              AGG ATC ATC ACA GAC AGC GAG AGT GAC ATC CTC TTC TTC                        TGG                                                                              GAA ACG CAA GGC AAT AAG AAC TAC TTC AAG TCA GCA GCC                        AAC                                                                              CCA CAG CTG TTT ATT GCC ACA AAG CCA GAA CAT CTG GTG                        CAC                                                                              ATG GCA AGG GGG TTA CCT TCG ATG ACT GAT TTT CAG ATC                        TCA(3')                                                                     [ B-1]                                                                        __________________________________________________________________________

                                      TABLE 4-1                                   __________________________________________________________________________    Met                                                                              Ala                                                                              Lys                                                                              Val                                                                              Pro                                                                              Asp                                                                              Leu                                                                              Phe                                                                              Glu                                                                              Asp                                                                              Leu                                                                              Lys                                                                              Asn                                                                              Cys                                                                              Phe                                 Ser                                                                              Glu                                                                              Asn                                                                              Glu                                                                              Glu                                                                              Tyr                                                                              Ser                                                                              Ser                                                                              Ala                                                                              Ile                                                                              Asp                                                                              His                                                                              Leu                                                                              Ser                                                                              Leu                                 Asn                                                                              Gln                                                                              Lys                                                                              Ser                                                                              Phe                                                                              Tyr                                                                              Asp                                                                              Ala                                                                              Ser                                                                              Tyr                                                                              Glu                                                                              Pro                                                                              Leu                                                                              His                                                                              Glu                                 Asp                                                                              Cys                                                                              Met                                                                              Asn                                                                              Lys                                                                              Val                                                                              Val                                                                              Ser                                                                              Leu                                                                              Ser                                                                              Thr                                                                              Ser                                                                              Glu                                                                              Thr                                                                              Ser                                 Val                                                                              Ser                                                                              Pro                                                                              Asn                                                                              Leu                                                                              Thr                                                                              Phe                                                                              Gln                                                                              Glu                                                                              Asn                                                                              Val                                                                              Val                                                                              Ala                                                                              Val                                                                              Thr                                 Ala                                                                              Ser                                                                              Gly                                                                              Lys                                                                              Ile                                                                              Leu                                                                              Lys                                                                              Lys                                                                              Arg                                                                              Arg                                                                              Leu                                                                              Ser                                                                              Leu                                                                              Asn                                                                              Gln                                 Pro                                                                              Ile                                                                              Thr                                                                              Asp                                                                              Val                                                                              Asp                                                                              Leu                                                                              Glu                                                                              Thr                                                                              Asn                                                                              Val                                                                              Ser                                                                              Asp                                                                              Pro                                                                              Glu                                 Glu                                                                              Gly                                                                              Ile                                                                              Ile                                                                              Lys                                                                              Pro                                                                              Arg                                                         [A-2]                                                                         __________________________________________________________________________

                                      TABLE 4-2                                   __________________________________________________________________________    (5')ATG                                                                            GCC AAA GTC CCT GAT CTG TTT GAA GAC CTA AAG AAC TGT                        TTC                                                                              AGT GAA AAT GAG GAA TAC AGC TCT GCC ATT GAC CAT CTT                        TCT                                                                              CTG AAT CAG AAA TCC TTC TAT GAT GCA AGC TAT GAA CCA                        CTT                                                                              CAT GAA GAC TGC ATG AAT AAA GTT GTG TCT CTG AGT ACC                        TCT                                                                              GAA ACC TCT GTG TCA CCC AAC CTC ACC TTC CAG GAG AAT                        GTG                                                                              GTG GCA GTA ACA GCC AGT GGG AAG ATT CTG AAG AAA AGA                        CGC                                                                              TTG AGT TTA AAC CAG CCC ATC ACG GAT GTT GAC CTG GAG                        ACC                                                                              AAT GTC AGT GAT CCA GAA GAA GGA ATC ATC AAG CCC AGA(3')                  [B-2]                                                                         __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    (human)                                                                       __________________________________________________________________________     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                    __________________________________________________________________________     [Note]:                                                                       1) ***means the stop codon.                                                   2) The bracket means the region coding for mature IL1.                   

    TABLE 6      (human)      ##STR30##      ##STR31##      ##STR32##      ##STR33##      ##STR34##      ##STR35##      ##STR36##      ##STR37##      ##STR38##      ##STR39##      ##STR40##      ##STR41##      ##STR42##     [Note]:     ***means the stop condon.

                                      TABLE 7                                     __________________________________________________________________________    (rabbit)                                                                      __________________________________________________________________________     ##STR43##                                                                     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                    __________________________________________________________________________     [Note]:                                                                       1) ***means the stop codon.                                                   2) The bracket means the region coding for mature IL1.                   

    TABLE 8-1      ##STR57##      ##STR58##      ##STR59##      ##STR60##      ##STR61##      ##STR62##      ##STR63##      ##STR64##      ##STR65##      ##STR66##      ##STR67##      ##STR68##     [Note]:     1) Upper sequence is the nucleotide sequence of a DNA encoding rabbit IL1     precursor.     2) Lower sequence is the nucleotide sequence of a DNA encoding human IL1     precursor.     3) -  - - means deletion.     4)*** means the stop codon.     ##STR69##

                                      TABLE 8-2                                   __________________________________________________________________________     ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                                                     ##STR73##                                                                     ##STR74##                                                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                     ##STR78##                                                                     ##STR79##                                                                     ##STR80##                                                                     ##STR81##                                                                    __________________________________________________________________________     [Note]:                                                                       1) Upper sequence is polypeptide of rabbit IL1 precursor.                     2) Lower sequence is polypeptide of human IL1 precursor.                      3) ---  means deletion.                                                       ##STR82##                                                                

What is claimed is:
 1. A method of treating sarcoma in an animal whichcomprises administering an isolated mature human interleukin 1αpolypeptide in an amount effective to treat said sarcoma wherein saidpolypeptide has an amino acid sequence selected from the groupconsisting of amino acids 113 to 271, 128 to 271, 113 to 267 and 128 to267 of FIG. 11.